Insect viruses and their uses in protecting plants

ABSTRACT

The present invention relates to an isolated small RNAvirus capable of infecting insect specifies including Heliothis species, and to the nucleotide sequences and proteins encoded thereby. The invention contemplates uses of the virus in controlling insect attach in plants.

This application is a continuation of U.S. Ser. No. 09/991,262, filedNov. 20, 2001, which is a continuation of U.S. Ser. No. 09/234,238 filedJan. 20, 1999, now abandoned, which is a continuation of U.S. Ser. No.08/440,522, filed May 12, 1995, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 08/089,372, filed Jul. 8, 1993,now abandoned, which claims the benefit of the filing date of AustralianProvisional Application Ser. No. PL 4801 filed on Aug. 14, 1992.

FIELD OF THE INVENTION

The present invention relates to insect viruses useful in control ofinsect attack on plants. It particularly relates to biologicalinsecticides, especially those comprised of insect viruses. Inparticular applications, the invention also provides recombinant virusesand transgenic plants.

BACKGROUND OF THE INVENTION

There is increasing awareness of the desirability of insect pest controlby biological agents. Considerable effort in recent years has beendevoted to the identification and exploitation of DNA viruses with largegenomes, especially the baculoviruses. These viruses generally requireextensive genetic manipulation to become effective insecticides, and thefirst such modified viruses are only now being evaluated.

In contrast, very little effort has been devoted to the study and use ofsmall viruses with RNA genomes.

Four main groups of small RNA viruses have been isolated from insects.These include members of the Picornaviridae, the Nodaviridae, theTetraviridae and the unclassified viruses. Descriptions of these groupscan be found in the Atlas of Invertebrate Viruses (eds J. R. Adams andJ. R. Bonami) (CRC Press, Boca Raton, 1991) and Viruses of Invertebrates(ed. E. Kurstak) (Marcel Dekker, New York, 1991). These disclosuresrelating to these viruses concern their pathology and biology, not theiruse in biological control.

Further information regarding small RNA viruses of insects an be foundin P. D. Scotti et al (1981) “The biology and ecology of strains of aninsect small RNA virus complex” Advances in Virus Research 26, 117-143.This review describes the insect picornaviruses cricket paralysis virusand Drosophila C virus (diameters estimated at 27-30 nm with one RNAcomponent of 7.5-8.5 kb). N. F. Moore & T. W. Tinsley (1982) The smallRNA viruses of insects. Brief review Archives of Virology 72, 229-245.This review included viruses of the following families:

Nodaviridae (diameter 29-30 nm, 2 RNA components totalling 4.5 kb)

Picornaviridae (diameter 27-30 nm, one RNA component of 7.5-8.5 kb)

Nudaurelia β family (now called Tetraviridae) (diameter around 35 nm,

-   -   either one RNA of 5.5 kb or two totalling 8 kb)

N. F. Moore, B. Reavy & L. A. King (1985) General characteristics, geneorganisation and expression of small RNA viruses of insects. Journal ofgeneral Virology 66, 647-659. This reference defines small RNA virusesof insects as being those less than 40 nm in diameter. The review coversPicornaviridae, Nodaviridae and the Nudaurelia_family (now calledTetraviridae).

D. Hendry, V. Hodgson, R Clark and J Newman (1985) Small RNA virusesco-infecting the pine emperor moth (Nudaurelia cytherea capensis).Journal of general Virology 66, 627-632 described viruses with meandiameters of 40 nm and 38 nm and one or two RNA components up to 5.5 kbin length.

Most recently, the term insect small RNA viruses has been used by one ofthe present inventors to cover three main recognised toxic groups: thePicornaviridae, the Tetraviridae and the Nodaviridae (P.Scotti &P.Christian (1994) The promises and potential problems of using smallRNA insect viruses for insect control. Sains Malaysiana 23, 9-18).

These references illustrate a long standing usage of the term in thisfield of the term “small RNA virus” for viruses with certaincharacteristics as listed above. Another important characteristic ofthese virus groups is that they are not occluded, in contrast to manylarge viruses like the cytoplasmic polyhydrosis (RNA) viruses or the DNAbaculoviruses, granulosis viruses and entomopox viruses. The term wouldalso be applied to viruses not members of the three families listedabove, as long as they satisfied the definition of being up to 40 nm insize. There are reports of such unclassified viruses (eg in Hendry etal. 1985). Moreover, the taxonomic status of some members of theTetraviridae still requires clarification and it might even be possiblefor this family to be split, with HaSV and other members with two RNAcomponents in their genome being separated from those with only onecomponent, like the type member Nudaurelia_virus, which has not yet beensequenced. The above definition of “small RNA virus” would still coverall members of such virus families.

SUMMARY OF THE INVENTION

In a first aspect of the present invention there is provided an isolatedsmall RNA virus wherein the virus is up to 40 nm in size, is notoccluded and infects insect species including Heliothis species.

In one particular embodiment, the present invention provides an isolatedpreparation of Heliothis armigera stunt virus referred to as “HaSV”herein.

In a further aspect of the present invention there is provided anisolated nucleic acid molecule comprising a nucleic acid sequencehybridizable with RNA 1 (SEQ ID No: 39) or RNA 2 (SEQ ID No: 47)described herein under low stringency conditions.

In still a further aspect the invention provides a vector comprising anucleic acid molecule, the sequence of which is hybridizable with RNA 1(SEQ ID No: 39) or RNA 2 (SEQ ID No: 47) as described herein. Thesevectors include expression and transfer vectors for use in animalsincluding insect, plant and bacterial cells.

In a further aspect the invention provides an isolated protein orpolypeptide preparation of the proteins or polypeptides derivable fromthe isolated virus of the present invention. The invention also extendsto antibodies specific for the protein and polypeptide preparations.

In a yet further aspect the invention provides a recombinant insectvirus vector incorporating all or a part of the isolated virus of thepresent invention.

In a still further aspect of the present invention there is provided amethod of controlling insect attack in a plant comprising geneticallymanipulating said plant so that it is capable of producing HaSV ormutants, derivatives or variants thereof or an insecticidally effectiveportion of HaSV, mutants, variants or derivatives thereof such thatinsects feeding on the plants are deleteriously effected. The presentinvention also provides a transgenic plant so manipulated.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 a is a restriction map of RNA 1 (SEQ ID No. 39) clones.

FIG. 1 b is a restriction map of RNA 2 (SEQ ID No. 47) clones.

FIG. 2 is the complete sequence of RNA 1 (SEQ ID No: 39) and of majorencoded polypeptides, namely replicase (SEQ ID No: 40), 11a (SEQ ID NO:42), 11b (SEQ ID NO: 44) and P14 (SEQ ID NO: 46).

FIG. 3 a is the complete sequence of RNA 2 (SEQ ID No. 47) in theauthentic version, and its encoded polypeptides, namely P17 (SEQ ID No:48) and P71 (SEQ ID No: 50).

FIG. 3 b is the sequence of RNA 2 variant (a SC version) (SEQ ID No: 51)and its major encoded polypeptide(s),namely P70 (SEQ ID No: 52).

FIGS. 4 a and 4 b illustrate bioassay data showing HaSV induced stuntingof larvae.

FIG. 5 is a map of Vector plasmid pT7T2b and PT7T2c.

FIG. 6 is a schematic representation of the proteins encoded by RNA 1(SEQ ID No. 39) and RNA 2 (SEQ ID No. 47).

FIG. 7 is a schematic representation of the proteins expressed by RNA 2(SEQ ID No. 47) in bacteria DNA fragments encoding P17 (SEQ ID No. 48),P71 (SEQ ID No. 50), P64, P7 and the fusion protein P70 (SEQ ID No. 52)were synthesized by PCR. The flanking Ndel and BamHI sites used incloning are indicated. (Note that P17 is followed by BgIII site, whosecohesive ends are compatible with those of BamHI).

FIG. 8 illustrates the 3′-terminal secondary structure of HaSV RNAs. ThetRNA-like structures at the 3′ ends of RNAs 1 and 2 (SEQ ID No. 39 & 47)are shown. Residues in bold are common to both sequences.

FIG. 9 Expression strategies for HaSV cDNAs in insect cells. The upperpart of the figure shows the genome organization of RNAs 1 and 2 (SEQ IDNos. 39 & 47). The lower part shows insertion of cDNAs corresponding tothese RNAs into a plasmid vector, between heat shock protein (HSP70)promoter of Drosophila and a suitable polyadenylation (pA) signal. TheHSP promoter was obtained by PCR using suitable primers, with a BamHIsite inserted by PCR immediately upstream of the start of thetranscription, giving the following sequence: GGATCCACAGnnn (SEQ ID No.1), where the underlined residue is the transcription start site foreither RNA. The cDNAs are terminated by Clal sites, allowing directlinkage to ribozyme sequences as described in the text.

FIG. 10 Ribozymes to yield correct 3′ ends. The sequences of ribozymesinserted as short cDNA fragments into HaSV cDNA clones are shown. Theribozyme fragments were assembled and cloned as described in the text.Designed self-cleavage points are indicated by bold arrows.

FIG. 11 Immunoblots to map epitopes on HaSV. A. Detected with HaSVantiserum. Lane 1: pTP70delSP; lane 2: pTP70; lane 3: pTP17; lane 4:control; lane 5: pTP70delN; lane 6: pTP70; lane 7: pTP71; lane 8: HaSVvirions; lane 9: molecular weight markers. B. Detected with HaSVantiserum. Lane 1: pTP70delN; lane 2: pTP70 delSPN; lane 3: pTP70. C.Detected with an antiserum to the Bt toxin (CryIA(c)). Lane 1: pTP70;lane 2: HaSV virions; lane 3: control extract.

FIG. 12 New field isolates of HaSV. The genomic organization of RNA 2 isshown at the top of the Figure. PCR using appropriate primers with BamHIrestriction sites and in some cases altered context sequences of the AUGinitiating translation of the P17 (SEQ ID No. 48) or P71 (SEQ ID No. 50)genes were used to make fragments for cloning into the BamHI sites ofthe expression vectors. Constructs 17E71 (SEQ ID No. 35) and P71 (SEQ IDNo. 50) have altered context sequences of the AUG initiating translationof the P 17 (SEQ ID No. 48) and P71 (SEQ ID No. 50) genes respectively;these alterations correspond to the context derived from the JHE gene(see text). All context sequences are given on the right of the Figure.R2 is a clone of the complete RNA sequence as a BarrHI fragment in thevector.

FIG. 13 Maps of the expression constructs in baculovirus vectors.

FIG. 14 a to e Various strategies utilizing the present invention.

FIG. 15 Expression of RNAs 1 and 2 (SEQ ID Nos. 39 and 47) frombaculovirus vectors. The full length cDNA clone of HaSV RNA 1 or 2

(SEQ ID Nos. 39 & 47) was inserted as a BamHI fragment into thebaculoexpression vectors. PCR. was used to add BamHI sites immediatelyadjacent to the 5′ and 3′ termini of the RNA 1 sequence; sequences ofthe primers are given in the text. Constructs R1RZ and R2RZ carrycis-acting ribozymes immediately adjacent to the 3′ end of the sequenceof RNA 1 and 2 (SEQ ID Nos. 39 & 47) respectively.

FIG. 16 Expression strategies for HaSV cDNAs in plant cells. The upperpart of the Figure shows the genome organization of RNAs 1 and 2 (SEQ IDNos. 39 & 47). The lower part shows insertion of cDNAs corresponding tothese RNAs into a plasmid vector, between 35S promoter of cauliflowermosaic virus and the polyadenylation (pA) signal on plasmid pDH51(Pietrzak et al., 1986). The cDNAs were obtained by PCR using suitableprimers, with a BaMHI site immediately upstream of the start of eachcDNA. The cDNAs are terminated by ClaI sites, allowing direct linkage toribozyme sequences as described in the text.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

A first aspect of the invention contemplates use of small RNA virusesfor biological control of insects. In particular, in accordance with thefirst aspect of this invention there is provided an isolated small RNAvirus, particularly H. armigera stunt virus or mutants, variants orderivatives thereof capable of infecting insects, in particular theinsect species such as Helicoverpa armigera. The small RNA virus isolateof the instant invention is insecticidal and in particular stunts thegrowth of insect larvae, for example Helicoverpa armigera larvae andinhibits or prevents development into the adult stage.

The small RNA viruses of the instant invention have insecticidal,anti-feeding, gut-binding or any synergistic property or other activityuseful for insect control.

In particular, Helicoverpa armigera stunt virus (HaSV) particles areisometric and approximately 36 nm in diameter with a buoyant density onCsCl gradients of 1.36 g/ml. The virus is composed of two major capsidproteins of approximately 64 and 7 KDa in size as determined onSDS-PAGE. The HaSV genome is much later than the largest known nodavirus(another class of RNA viruses) and comprises two ss (+) RNA molecules ofapproximately 5.3 and 2.4 kb. The genome appears to lack a blockage ofunknown structure at the 3′ termini that is found in Nodaviridae. TheHaSV genome however shares a capped structure and non-polyadenylationwith Nodaviridae. HaSV differs significantly from Nodaviridae andNudaurelia w virus in terms of its immunological properties. Inparticular the large capsid protein has different antigenicdeterminants. Other properties of HaSV are described in the Examples.

The host range of HaSV includes Lepidopterans such as from the subfamilyHeliothinae. Species known to be hosts are Helicoverpa (Heliothis)armigera,

H. punctigera, H. zea, Heliothis virescens and other such noctuides asSpodoptera exigua. H. armigera which is known by the common names cornear worm, cotton ball worm, tomato grub and tobacco bud worm is a pestof economic significance in most countries. H.punctigera, the native budworm, is a pests of the great economic significance in Australia.Members of the Heliothinae, which include Helicoverpa and Heliothis, andespecially H.armigera are among the most important and widespread pestsin the world. In the US Heliothis virescens and Helicoverpa zea areparticularly important pests.

The first aspect of the invention provides an isolated small RNA viruscapable of infecting insects including Heliothis species. In aparticularly preferred form the invention relates to mutants, variantsand derivatives of HaSV. The terms “mutant”, “variant and “derivative”include all naturally occurring and artificially created viruses orviral components which differ from the HaSV isolate as herein describedin nucleotide content or sequence, amino acid content or sequence,immunological reactivity, non-glycosylation or glycosylation patternand/or infectivity but generally retain insecticidal activity.Specifically the terms “mutant”, “variant” and “derivative” of HaSVcovers small RNA viruses which have one or more functionalcharacteristic of HaSV described herein. Examples of mutants, variantsor derivatives of HaSV include small RNA viruses that have differentnucleic or amino acid sequences from HaSV but retain one of morefunctional features of HaSV. These may include strains with geneticallysilent substitutions, strains carrying replication and encapsidationsequences and signals that are functionally related to HaSV, or strainsthat carry functionally related protein domains.

In a preferred aspect the invention relates to mutants, variants orderivatives 2 of HaSV which encode replication or encapsidationsequences, structures or signals with 60%, preferably 70%, morepreferably 80%, still more preferably 90% and even more preferably 95%nucleotide sequence identity to the nucleotide sequences HaSV.

In another preferred aspect the invention relates to mutants, variantsor derivatives of HaSV which encode proteins with at least 50%,preferably 60%, preferably 70%, more preferably 80%, still morepreferably 90% and even more preferably 95% amino acid sequence identityto proteins or polypeptides of HaSV.

In another preferred aspect the invention relates to mutants, variantsor derivatives of HaSV with 50%, more preferably 60%, still morepreferably 70%, more preferably 80%, still more preferably 90 or 95%nucleotide sequence identity to the following biologically activedomains encoded by the HaSV genome:

RNA 1 (SEQ ID No: 39)—amino acid residues 401 to 600 or the otherdomains in the replicase

RNA 2 (SEQ ID No: 47) (in the capsid protein)

amino acid residues 273 to 435

amino acid residues 50 to 272

amino acid residues 436 to the COOH terminus

Preferably the viral isolate of the present invention is biologicallypure which means a preparation of the virus comprising at least 20%relative to other components as determined by weight, viral activity orany other convenient means. More preferably the isolates are 50% pure,still more preferably it is 60%, even more preferably it is 70% pure,still more preferably it is 80% pure and even more preferably it is 90%or more, pure.

In a second aspect the present invention relates to a nucleotidesequence or sequences hybridizable with those of HaSV. The termnucleotide sequence used herein includes RNA, DNA, cDNA and nucleotidesequences complementary thereto. Such nucleotide sequences also includesingle or double stranded nucleic acid molecules and linear andcovalently closed circular molecules. The nucleic acid sequences may bethe same as the HaSV sequences as herein described or may contain singleor multiple nucleotide substitutions and/or deletions and/or additionsthereto. The term nucleotide sequence also includes sequences withsufficient homology to hybridize with the nucleotide sequence under low,preferably medium and most preferably high stringency conditions(Sambrook J, Fritsch, E. F. & Maniatis T. (1989) Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbour Laboratories Press)and to nucleotide sequences encoding functionally equivalent sequences.In still a more preferred embodiment the invention comprises thenucleotide sequences of genome components 1 and 2 (SEQ ID Nos: 39 and47) as represented by FIGS. 1 and 2 hereinafter or parts thereof, ormutants, variants, or derivatives thereof. The terms “mutants”,“variants” or “derivatives” of nucleotide genome components 1 and 2 (SEQID Nos: 39 and 47) has the same meaning, when applied to nucleotidesequences as that given above and includes parts of genome components 1and 2 (SEQ ID Nos: 39 and 47).

The second aspect of the invention also relates to nucleotide signals,sequences or structures which enable the nucleic acid on which they arepresent to be replicated by HaSV replicase. Furthermore the inventionrelates to the nucleotide signals, sequences or structures which enablenucleic acids on which they are present to be encapsidated.

In a particularly preferred embodiment of the second aspect, theinvention comprises nucleotide sequences which are mutants of the capsidgene having the following sequences: ATG GGC GAT GCC GGC GTC GCGT TCACAG (SEQ ID No: 2) ATG GAG GAT GCT GGA GTG GCG TCA CAG (SEQ ID No: 3)ATG AGC GAG GCC GGC GTC GCG TCA CAG (SEQ ID No: 4)

In a preferred aspect the invention relates to nucleotide sequences ofHaSV encoding insecticidal activity including the capsid protein geneand P17 (SEQ ID No: 48) and mutants, variants and derivatives thereof.

In another preferred aspect the invention comprises nucleotide sequencesincluding the following ribozyme oligonucleotides: (SEQ ID No: 5)5′ CCATCGATGCCGGACTGGTATCCCAGGGGG (called “HVR1Cla” herein) (SEQ ID No:6) 5′ CCATCGATGCCGGACTGGTATCCCGAGGGAC (called “5′HVR2Cla” herein) (SEQID No: 7) 5′ CCATCGATGATCCAGCCTCCTCGCGGCGCCGGATGGGCA (called“RZHDV1” herein) (SEQ ID No: 8)5′ GCTCTAGATCCATTCGCCATCCGAAGATGCCCATCCGGC (called “RZHDV2” herein) (SEQID No: 9) 5′ CCATCGATTTATGCCGAGAAGGTAACCAGAGAAACACAC (called“RZHC1” herein) (SEQ ID No: 10)5′ GCTCTAGACCAGGTAATATACCACAACGTGTGTTTCTCT (called “RZHC2” herein)

Ribozyme sequences are useful for obtaining translation, replication andencapsidation of the transcript. It is therefore desirable to cleave thetranscript downstream of its t-RNA-like structure or poly A tail priorto translation, replication or encapsidation of the transcript.

The present invention also further extends to oligonucleotide primersfor the above sequences, antisense sequences and nucleotide probes forthe above sequences and homologues and analogues of said primers,antisense sequences and probes. Such primers and probes are useful inthe identification, isolation and/or cloning of genes encodinginsecticidally effective proteins or proteins required for viralactivity, from HaSV or another virus (whether related or unrelated)carrying a similar gene or similar RNA sequence. They are also useful inscreening for HaSV or other viruses in the field or in identifying HaSVor other viruses in insects, especially in order to identify relatedviruses capable of causing pathogenecity similar to HaSV.

Any pair of oligonucleotide primers derived from either RNA 1 or RNA 2(SEQ ID Nos: 39 and 47) and located between ca 300 and 1500 bp apart canbe used as primers. The following pairs of primer sequences exemplifyparticularly preferred embodiments of the present invention:Specifically for RNA 1 (SEQ ID No: 39):

1 HVR1B5′ (SEQ ID No: 38) (described below) and the primer complementaryto nucleotides 1192-1212 of FIG. 1. 2. The primer corresponding tonucleotides 4084 and 4100 of FIG. 13 and the primer HVR13p (SEQ ID No:12) described below

Specifically for RNA 2 (SEQ ID No: 47):

1. The primer corresponding to nucleotides 459 to 476 of FIG. 2 and theprimer complementary to nucleotides 1653 to 1669 of FIG. 2 (this wouldinclude the central variable domain) 2. R2cdha5 and the primercomplementary to nucleotides 1156 to 1172of FIG. 2

3. The primer corresponding to nucleotides 1178 to 1194 and the primercomplementary to nucleotides 2072 to 2091 (of FIG. 2).

Other combinations giving shorter fragments are also possible.

Further preferred primers include: (SEQ ID No: 11)5′ GGGGGGAATTCATTTAGGTGACACTATAGTTCTGCCTCCCCGGAC (called“HvR1SP5p” herein) (SEQ ID No: 12) 5′ GGGGGGATCCTGGTATCCCAGGGGGGC(called “HvR13p” herein) (SEQ ID No: 13) 5′ CCGGAAGCTTGTTTTTCTTTCTTTACCA(called “Hr2cdna5” herein) (SEQ ID No: 14)5′ GGGGGATCCGATGGTATCCCGAGGGACGCTCAGCAGGTGGCATAGG (called“HvR23p” herein) (SEQ ID No: 15)AAATAATTTTGTTACTTTAGAAGGAGATATACATATGAGCGAGCGAGCAC AC (called“HVPET65N” herein) (SEQ ID No: 16)AAATAATTTTGTTTAACCTTAAGAAGGAGATCTACATATGCTGGAGTGGC GTCAC (called“HVPET63N” herein) (SEQ ID No: 17) GGAGATCTACATATGGGAGATGCTGGAGTG(called “HVPET64N” herein) (SEQ ID No: 18) GTAGCGAACGTCGAGAA (called“HVRNA2F3” herein) (SEQ ID No: 19) GGGGGATCCTCAGTTGTCAGTGGCGGGGTAG(called “HVP65C” herein) (SEQ ID No: 20) GGGGATCCCTAATTGGCACGAGCGGCGC(called “HVP6C2” herein) (SEQ ID No: 21) AATTACATATGGCGGCCGCCGTTTCTGCC(called “HVP6MA” herein) (SEQ ID No: 22) AATTACATATGTTCGCGGCCGCCGTTTCT(called “HVP6MF” herein)

The invention also relates to vectors encoding the nucleotide sequencedescribed above and to host cells including the same. Preferably thesevectors are capable of expression in animal, plant or bacterial cell orare capable of transferring the sequences of the present invention tothe genome of other organisms such as plants. More preferably they arecapable of expression in insect and crop plant cells.

In a preferred aspect the invention relates to the vectors pDHVR1,pDHVR1RZ, pDHVR2, pDHVR2RZ, p17V71, pI7E71, pPH, pV71, p17V64, p17E64,pP64, pV64, pBacHVR1, pBacHVR1RZ, pBacHUR2, pBacHVR2RZ, pHSPR1,pHSPR1RZ, pHSPR2, pHSPR2RZ, pSR1(E3)A, pSR1(E3)B, pSR2A, pSR2B, pSX2P70,pSXR2P70, pSRP2B, pBHVR1B, pBHVR2B, pT7T2P64, pSR2P70, pT7T2P65,pT7T2P70, pT7T2-P71, pBSKSE3, pBSR15, pBSR25p, pSR25, phr236P70,phr235P65, pGemP63N, pGemP64N, pGemP65N, pP64N, pP65H, pTP6MA, pTP6MF,pTP17, pTP17delBB, pP656 or p70G as described hereinafter.

In a third aspect the invention relates to polypeptides or proteinsencoded by HaSV and to homologues and analogues thereof. This aspect ofthe invention also relates to derivatives and variants of thepolypeptides and proteins of HaSV. Such derivatives and variants includesubstitutions and/or deletions of one or more amino acids, and amino andcarboxy terminal fusions with other polypeptides or proteins. In apreferred aspect the invention relates to the proteins P7, P16, P17 (SEQID No: 48), P64, P70 (SEQ ID No: 52), P71 (SEQ ID No: 50), P11a (SEQ IDNo: 42), P11b (SEQ ID No: 44), P14 (SEQ ID No: 46) and P187 (SEQ ID No:40) described herein and to homologues and analogues thereof, includingfusion proteins particularly of P71 (SEQ ID No: 50) such as P70 (SEQ IDNo: 52) described herein. In a most preferred aspect the inventionrelates to polypeptides or proteins from HaSV which have insecticidalactivity themselves or provide target specificity for insecticidalagents. In particular the invention relates to polypeptides or fragmentsthereof with insect gut binding specificity, particularly to thevariable domains thereof as herein described. In addition, homologuesand analogues with said insecticidal activity of the polypeptides andproteins are also included within the scope of the invention. Inaddition the invention also relates to antibodies (such as monoclonal orpolyclonal antibodies or chimeric antibodies including phage antibodiesproduced in bacteria) specific for said polypeptide and proteinsequences. Such antibodies are useful in detecting HaSV and relatedviruses or the protein products thereof.

In a fourth aspect the invention provides an infectious, recombinantinsect virus including a vector, an expressible nucleic acid sequencecomprising all of, or a portion of the HaSV genome, including aninsecticidally effective portion of the genome and optionally, materialderived from another insect virus species or isolate(s).

Insect virus vectors suitable for the invention according to thisaspect, include baculoviruses, entomopoxviruses and cytoplasmicpolyhedrosis viruses. Most preferably, the insect virus vector isselected from the group comprising the baculovirus genera of nuclearpolyhedrosis viruses (NPV's) and granulosis viruses (GV's). In thisaspect of the invention the vector acts as a carrier for the HaSV genesencoding insectidical activity. The recombinant insect virus vector maybe grown by either established procedures Shieh, (1989), Vlak (in press)or any other suitable procedure and the virus disseminated as needed.The insect virus vectors may be those described in copendingInternational application No. PCT/AU92/00413.

The nucleic acid sequence or sequences incorporated into the recombinantvector may be a cDNA, DNA or DNA sequence and may comprise the genome orportion thereof of a DNA or RNA of HaSV or another species. The term“material derived from another insect virus species or isolate” includesany nucleic acid sequence, or protein sequence or parts thereof whichare useful in exerting an insecticidal effect when incorporated in therecombinant vector of the invention. Suitable nucleic acid sequences forincorporation into the recombinant vector include insecticidallyeffective agents such as a neurotoxin from the mite Pyemotes tritici(Tomalski, M. D. & Miller, L. K. Nature 352, 82-85 (1991) a toxincomponent of the venom of the North African scorpion Androctonusaustralia Maeda, S. et al. Virology 184-777-780 (1991) Stewart, L. M. D.et al., Nature 352, 85-88 (1991), Conotoxins from the venom of Conusspp. (Olivera B. M. et al., Science 249, 257-263 (1990); Woodward S. R.et al., EMBO J. 9, 1015-1020 (1990); Olivera B. M. et al., Eur. J.Biochem. 202, 589-595 (1991).

The exogenous nucleic acid sequence may be operably placed into theinsect virus vector between a viral or cellular promoter and apolyadenylation signal. Upon infection of an insect cell, the vectorvirus will cause the production of either infectious virus genomic RNAor infectious encapsidated viral particles.

The promoters may be constitutively expressed or inducible. Theseinclude tissue specific promoters, temperature sensitive promoters orpromoters which are activated when the insect feeds on a metabolite inthe plant that it is desired to protect.

Recombinant insect virus vectors according to the present invention mayinclude nucleic acid sequences comprising all or an infectious orinsecticidally effective portion of genome the HaSV and optionallyanother insect virus species or isolate.

In a particularly preferred embodiment of the present invention there isprovided assembled capsids comprising one or more of the capsid proteinsof the present invention, or derivatives or variants thereof ascontemplated or described herein. These assembled virus capsids areuseful as vectors for insecticidal agents. As such the assembled viralcapsids may be used to administer insecticidal agents such as variousnucleotide sequences with insecticidal activity or various toxins to aninsect. Nucleotide sequences in the form of RNA or DNA which can be usedinclude those of the HaSV genome or other insect viruses. Toxins whichcan be used advantageously include those which are activeintracellularly and may also include neurotoxins with an appropriatetransportation mechanism to reach the insect neurones.

The efficacy or insecticidal activity of infectious genomic RNA or viralparticles produced by insect cells infected with insect vectorsaccording to this aspect of the invention, may be enhanced as describedbelow. Moreover the virus vector itself may include within a non-essential region(s), one or more nucleic acid sequences encodingsubstances that are deleterious to insects such as the insecticidallyeffective agents described above. Alternatively an extra genomecomponent may be added to the HaSV genome either by insertion into oneof the HaSV genes or by adding it to the ends of the genome.

In a particularly preferred embodiment there is provided a recombinantbaculovirus vector comprising HaSV or part thereof having insecticidalproperties.

Other modifications which may be made to the infectious recombinantinsect virus according to the fourth aspect include:

i) splitting the exogenous HaSV nucleic acid molecules comprising thegenome and cloning the fragments into the insect vector so that theycannot rejoin. One component, preferably the virus RNA replicase, couldbe expressed from a separately-transcribed fragment, the transcripts ofwhich would not be replicated by the replicase they encode. Theremainder of the genome (having insecticidal activity or encoding thecapsid protein or a separate toxin m-RNA) could be encoded by, forexample, a second separately-transcribed fragment, the transcripts ofwhich are capable of being amplified by the replicase. Consequently,whilst the transcripts from the second or other fragment would effecttheir insecticidal activity upon the infected insect cell, they wouldnot be able to infect another insect cell, (even if encapsidated)because the replicase or replicase-encoding transcripts would be absent;

This modification would allow an inherent biological containment to bebuilt into the insecticidal vectors, which, when used in conjunctionwith the use of non-persistent DNA virus vectors such as those describedin the above mentioned copending application, would allow a new level ofenvironmental safety greatly extending earlier approaches based onbaculovirus vectors.

ii) Manipulation of encapsidation signals or sequences essential forreplicase binding or production of sub-genomic mRNA's includingexpression of exogeneous insect control factors as RNAs dependent on thevirus for replication. This involves determination of RNA sequences andsignals important for replication and encapsidation of virus RNAs, suchas by analysis of replication of deletion mutants carrying reportergenes in appropriate cells, followed by studies on the transmission ofthe reporter gene to larvae by feeding of virus. These deletion mutantscan be used to carry genes for insect control factors/toxins to larvaeafter replacing the reporter gene by a suitable toxin gene such as shownin FIG. 12;

iii) using an insect promoter responsive to virus infection and, forexample, placing copies of the viral replicase gene under the control oftwo promoters, one which is constitutive or expressed at early stages ofvector infection, and the other being a cellular promoter turned on bythe ensuing RNA viral infection. This system would then make more copiesof the replicase MRNA available as the amount of its template increased.Such a promoter may be isolated using techniques analogous to enhancertrapping, that is, transforming insect cells with a suitable reportergene and looking for induction of the reporter upon virus infection of apopulation of transformed cells.

In a fifth aspect the invention relates to a method of controllinginsect attack in plants by genetically manipulating plants to expressHaSV or parts thereof which can confer insecticidal activity optionallyin combination with other insecticidally effective agents. Such plantsare referred to as transgenic plants.

The term “express” should be understood as referring to the process oftranscribing the genome or portion thereof into RNA or, alternatively,the process of transcribing the genome or portion thereof into RNA andthen, in turn, translating the RNA into a protein or peptide.

In a sixth aspect the invention relates to the transgenic plants per seas described above. Transgenic plants according to the invention may beprepared for example by introducing a DNA construct including a cDNA orDNA fragment encoding all or a desired infectious portion of HaSV, intothe genome of a plant. The cDNA or DNA fragment may, preferably, beoperably placed between a plant promoter and a polyadenylation signal.Promoters may cause constitutive or inducible expression of thesequences under their control. Furthermore they may be specific tocertain tissues, such as the leaves of a plant where insect attackoccurs but not to other parts of the plant such as that used for food.The inducible promoters may be induced by stimuli such as disturbance ofwind or insect movement on the plant's tissues, or may be specificallyturned on by insect damage to plant tissues. Heat may also be a stimulusfor promoter induction such as in spring where temperatures increase andlikelihood of insect attack also increases. Other stimuli such asspraying by a chemical (for instances a harmless chemical) may inducethe promoter.

The cDNA or DNA fragment may encode all or a desired infectious portionof the wild-type, recombinant or otherwise mutated HaSV. For example,deletion mutants could be used which lack segments of the viral genomewhich are non-essential for replication or perhaps pathogenicity.

The nucleotide sequences of the invention can be inserted into a plantgenome by already established techniques, for example by anAgrobacterium transfer system or by electroporation.

Plants which may be used in this aspect of the invention include plantsof both economic and scientific interest. Such plants may be those ingeneral which need protection against the insect pests discussed hereinand in particular include tomato, potato, corn, cotton, field pea andtobacco.

To enhance the efficacy of infectious genomic RNA or viral particlesexpressed by transgenic plants according to the invention, the DNAconstruct introduced into the plants′ genome may be engineered toinclude one or more exogenous nucleic acid sequences encoding substancesthat are deleterious to insects. Such substances include, for example,Bacillus thuringiensis d-toxin, insect neurohormones, insecticidalcompounds form wasp or scorpion venom or of heterologous origin, orfactors designed to attack and kill infected cells in such a way so asto cause pathogenesis in the infected tissue (for example, a ribozymetargeted against an essential cellular function).

DNA constructs may also be provided which include:

i) mechanisms for regulating pathogen expression (for example,mechanisms which restrict the expression of ribozymes to the insectcells) by tying for example, expression to abundant virus replication,production of minus-strand RNA or sub-genomic mRNA's; and/or

-   -   ii) mechanisms similar to, or analogous to, those described in        copending International patent application number PCT/AU92/00413        so as to achieve a limited-spread system (such as control of        replication).

Transgenic plants according to the present invention may also be capableof expressing all or an infectious or insecticidal portion of genomesfrom HaSV and one or more species or isolates of insect viruses.

In a seventh aspect of the invention HaSV, or insecticidally effectiveparts thereof, or the infectious recombinant virus vectors of the fourthaspect of the present invention may be applied directly to the plant tocontrol insect attack. HaSV or the recombinant virus vectors may beproduced either in whole or in part in either whole insects or inculture cells of insects or in bacteria or in yeast or in some otherexpression system. HaSV or the recombinant virus forms may be applied ina crude form, semi purified or purified form optionally in admixturewith agriculturally acceptable carrier to the crop in need ofprotection. HaSV may also be applied as a facilitator of infection whereexisting insect populations already infected with another agent, such asone or more other viruses whereby HaSV is able to act synergistically tobring about an insecticidal effect. Alternatively HaSV and another agentsuch as one or more viruses may be applied together to plants to controlinsects feeding thereon.

A deposit of HaSV No. 18.4 was made on Aug. 5th 1992 at the AustralianGovernment Analytical Laboratories. The deposit was given accession No.N92/35575.

EXAMPLE 1 Taxanomic, Physiochemical and Biochemical Characteristion ofan Insect Virus: HaSV

Materials and Methods

A Animals and Virus Production.

H. Armigara larvae were raised as described in Teakle R. E. and JensenJ. M. (1985) Heliothis punctiger in Singh P and Moore R. F. (eds)Handbook of Insect Rearing Vol 2., Elsevier, Amsterdam pp 313-322.Larvae were infected for virus production by feeding five day old larvaeon 10 mg pieces of diet to which 0.064 OD₂₆₀ units of HaSV had beenapplied. After 24 hours the larvae were then transferred to covered12-well plates (BioScientific, Sydney, Australia) that containedsufficient diet and grown for eight days after which they were collectedand frozen at −80

C until further processed. Frozen larvae were weighed to 100 g, placedinto 200 ml of 50 mM Tris buffer (pH 7.4), homogenized, and filteredthrough four layers of muslin. This homogenate was centrifuged in aSorvall SS-34 rotor at 10,000×g for 30 minutes whereupon the supernatantwas transferred to fresh tubes and recentrifuged in Beckman SW-28 rotorat 100K×g for 3 hours. The resultant band was collected and repelletedin 50 mM pH 7.2 Tris buffer in a Beckman SW-28 tube by centrifugation at100K×g for 3 hours. The pelleted virus was resuspended overnight in 1 mlof buffer at 4

C then layered onto a discontinuous CsCl gradient containing equalvolumes of 60% and 30% CsCl (w/v) in a Beckman SW-41 tube andcentrifuged at 12 h at 200×g. The resultant pellet was suspended in 100ml of buffer and frozen for further use.

B Particle Characterization.

Staining with acridine orange was as described in Mayor H. D. and HillN. O. (1961) Virology 14: p264. Buoyant density was estimated in CsClgradients according to Scotti P. D., Longworth J. F., Plus N, Crozier G.and Reignanum C. (1981) Advances in Virus Research 26: 117-143.

C Immunological Procedure.

Rabbit anti-sera to HaSV was produced by standard immunologicalprocedures. Rabbit antisera to the Nudaurelia o virus in addition to thevirus itself was provided by Don Hendry (Rhodes University, Grahamstown,South Africa). Rabbit antisera to the Nudaurelia b virus was supplied bythe late Carl Reinganum (Plant Research Institute, Burnley, Vic,Australia). The immunological relationship to the Nudaurelia w virus wasdetermined by the standard reciprocal double diffusion technique.Immunoblotting was performed according to Towbin H., Staeheln T. andGordon J. (1979) PNAS. Antibodies monospecific for the major 65 kDacapsid protein were prepared by incubating polyclonal antisera withsections of nitrocellulose blotted with the 65 kDa protein. Afterextensive washing in Tris buffered saline, the bound antibodies wereeluted in 5OmM citric buffer, pH 8.0 after a 5 minute incubation.

D Protein Characterization.

Polyacrylamide gel electrophoresis in the presence of SDS followed theprocedure of Laemmli UK 1970 Nature 227: 680-685 and was done with 12.5%gels unless otherwise noted with low and high molecular weight standardsfrom BioRad. Staining was done with a colloidal preparation of CoomassieBlue G-250 (Gradipore Ltd, Pyrmont, New South Wales, Australia).Determination of the M_(r) of the smallest protein was done with a 16%gel and standards of 3.4 kDa, 12.5 kDa and 21.5 kDa (BoehringerMannheim). Glycosylation of the viral proteins was determined by ageneral glycan staining procedure with reagents supplied by BoehringerMannheim; the positive control was fetuin. N-termini of proteins weresequenced using procedures described by Matsudairia (1989) Purificationof Proteins and Peptides by SDS-PAGE in A Practical Guide to Protein andPeptide Purification for Microsequencing ed Matsudaira P.T. AcademicPress, San Diego pp 52-72 on an Applied Biosystems 477A gas phasesequencer.

E Nucleic Acid Characterization.

RNA was removed from capsids by twice vortexing a virus suspension withequal volumes of neutralized phenol then with phenol/chloroformn(50:50). RNA was then precipitated from the aqueous phase in thepresence of 300 mM sodium acetate and 2.5 volumes of ethanol. Digestionsof the HaSV nucleic acid with RNAse A and DNAse I (Boehringer Mannheim)were done with pBSSK(−) phagemid ssDNA and dsDNA (Stratagene) and RNAcontrols (BRL). Denaturing agarose gel electrophoresis in the presenceof formaldehyde was performed according to Sambrook et al (1989). Thestate of polyandenylation of the viral RNA was determnined by twomethods. The first method was to compare the binding of identicalamounts (20 mg) of viral RNA and poly(A)-selected RNA from Helicoverpavirescens to a 1 ml slurry of 5mg of oligo-d(T) cellulose (Pharmacia) ina binding buffer consisting of 20 mM Tris pH 7.8, 500 mM NaCl, 1 mM EDTAand 0.04% SDS. The second method was to observe specific priming ofviral RNA and viral RNA polyadenylated with poly(A) polymerase(Pharmacia) with d(T) ₁₆A/C/G primers in RNA sequencing reactions usingreverse transcriptase (US Biochemical) and a protocol provided by thesupplier. The 5′ cap structure of the genomic RNA and HaSV wasdetermined by observing the ability of polynucleotide kinase tophosphorylate viral RNA with and without preincubation with tobacco acidpyrophosphatase and alkaline phosphatase (Promega) under conditionsdescribed by the supplier.

F In Vitro Translation of HaSV RNA.

In vitro translation of HaSV RNA was performed with lysates of bothrabbit reticulocytes and wheat germ (Promega) as directed by thesupplier. Reactions were conducted in 10 ml volumes with 1.0 mg of RNAin the presence of five u Ci ³⁵S-methionine. The labelled proteins wereresolved on 10% and 14% SDS-PAGE gels as described above then visualisedby autoradiography of the dried gels. The two viral RNAs were separatedby a “freeze and squeeze” method after resolution on nondenaturinglow-melting-point agarose gels in TAE (Sambrook, et al. 1989). Briefly,agarose slices containing the RNA were melted at 65

C in a volume of TAE buffer equal to six times the agarose volume. Thesolution was allowed to gel on ice before freezing at −80

C for 30 minutes. The frozen solution was thawed on ice then centrifugedat 14,500×g for 10 minutes after which the supernatant was withdrawn andprecipitated by the addition of ethanol.

G Bioassay of Virus-Induced Pathogensis

Known amounts of virus isolate, as shown in FIG. 3, were fed to larvaeat the growth stages indicated by admixture to stadnard diet. At thetime points shown, the larvae were weighed and the mean and SDcalculated. Growth of infected larvae was compared to those ofuninfected control populations from the same hatching batch in everyexperiment.

Results

i) Characteristics and Taxonomy of HaSV

The virus particles are isometric and are approximately 36-38 nm indiameter. They are composed of two major capsid proteins, of 65 kDa and6 kD is size. The virions contain two single-stranded (+) RNA species of5.3 kb and 2.4 kb length. The virus bears a similarity in these respectsto the Nudaurelia w virus, which has been tentatively regarded as amember of the Tetraviridae; these two viruses differ however, in theabove respects from other viruses in this group and are likely to formna new virus family, sharing chiefly their capsid structure (T=4) withthe Tetraviridae.

ii) Particle Characterization and Serology.

The buoyant density of HaSV was calculated to be 1.296 g/ml in CsCI atpH 7.2. The A₂₆₀/A₂₈₀ ratio of HaSV viral particles was 1.22 indicatinga nucleic acid content of approximately 7% (Gibbs and Harrison, (1976)Plant Virology: The Principles London: Edward Arnold. Reciprocalimmuno-double diffusion comparisons between HaSV and the Nudaurelia wvirus showed no serological relationship. The more sensitive techniqueof immunoblotting also showed a complete lack of any antigenicrelationship. In addition, HaSV did not react with antisera to theNudaurelia b virus in a immuno-diffusion test or when immunoblotted.However, no Nudaurelia b virus was available as a positive control inthese latter two immunological experiments. When HaSV was stained withacridine orange then irradiated with 310 nm UV light, the particlesfluoresced red which indicated a single stranded genome.

iii) Protein Characterization.

Examination of the capsid proteins of HaSV with polyacrylamide gelelectrophoresis in the presence of SDS showed variable results dependingon the quantity of protein present. At low protein loadings, twoproteins in major abundance were evident that had M_(r)'s of 65,000 and6,000 along with a protein in minor abundance with M_(r) of 72,000 (datanot shown). When more protein was present on the gels, however, at least12 more distinct bands with M_(r)'s ranging between 15,000 and 62,000became evident. Probing the resolved and blotted proteins withantibodies monospecific for the major 65 kDa capsid protein showed allbut two of the proteins shared common antigens with the major 65 kDaprotein. The major 6 kDa capsid protein and a minor band migrating atM_(r)=16,000 failed to react with both the monospecific antibodies anduntreated antisera.

The capsid proteins were shown to be non-glycosylated as they failed toreact with a hydrazine analog after oxidation with periodic acid. TheN-terminus of the 65 kDa protein appeared to be blocked in some manneras two efforts to conduct an Edman degradation failed. After the secondattempt, the sample was treated with n-chlorosuccinimide and shown to bein a quantity normnally adequate for sequencing. The N-terminus of the 6kDa protein, however, was not blocked as an unambiguous 16-residuesequence was readily obtained. The sequence of the N-terminus of the 6kDa capsid protein and those of a cyanogen bromide cleaved fragment ofthe 65 kDa protein are as follows: 6 kDa protein: (SEQ ID No: 23)PheAlaAlaAlaValSerAlaPheAlaAlaAsnMetLeuSerSerValLeuLysSer 65 kDaprotein: (SEQ ID No: 24)ProThrLeuValAspGlnGlyPheTrpIleGlyGlyGlnTyrAlaLeuThrProThrSer

Detailed sequence analysis of the RNA genome carried out in Example 3showed that RNA 1 (SEQ ID No: 39) encodes a protein of molecular weight186,980 hereinafterreferred to as P187 (SEQ ID No: 40) and RNA 2 (SEQ IDNo: 47) encodes proteins with molecular weight 16, 522 (called P17 (SEQID No: 48)) and 70,670 (called P71 (SEQ ID No: 50)). P71 (SEQ ID No: 50)is processed into two proteins of molecular weight 63,378 (called P64)and 7,309 (called P7).

iv) Nucleic Acid Characterization

The extracted nucleic acid from HaSV was readily hydrolysed by RNAse Abut not by DNAse 1. Denaturing agarose gel electrophoresis of theextracted RNA genome of HaSV indicated two strands that migrated at 5.5kb and 2.4 kb. The RNA strands were shown not to have extensive regionsof polyadenylation as only 24% of the viral RNA bound to the oligo-d(T)cellulose matrix as opposed to 82% of poly(A)-selected RNA. Furtherevidence for the non-polyadenylation of the viral genome was provided bythe observation that the oligo primer, d(T) ₁₆G, gave a clear sequencingladder using reverse transcriptase only after in vitro polyadenylationof the viral strands with poly(A)-polymerase.

The demonstration that the strands could be modified withpoly(A)-polymerase also showed the lack of any 3′ modification. The 5′termini of the viral strands were shown to be capped, most likely withm⁷G(5′)ppp(5′)G, as they could not be labelled with polynucleotidekinase unless pretreated with tobacco acid pyrophosphatase and alkalinephosphatase.

v) In Vitro Translation.

In vitro translation of the viral RNA yielded different results in thetwo translation systems used (data not shown). The 5.5 kb RNA translatedvery poorly in the reticulocyte system whereas it produced in thewheatgerm system more than 20 proteins ranging in size fromM_(r)=195,000 to M_(r)=12,000. The 2.4 kb viral RNA strand yielded amajor protein with an M_(r)=24,000 in both systems in addition to aminor protein at M_(r)=70 kDa. A time course of the translation reactionwith the 5.5 kb RNA strand showed all labelled proteins were produced atsimilar rates indicating that the smaller products did not arise throughprocessing of the larger ones. However when a time course experiment wasdone with translation of the smaller 2.4 kb RNA strand, the 24 kDaprotein appeared before the 70 kDa protein.

vi) Presence of Another Form of HaSV

Frequently, during purification of HaSV virions, a minor band appearedin varying amounts on the CsCl gradient that had a buoyant density of1.3 g/ml. On four occasions, when particles from this minor band wereused to infect H. armigera larvae that were then processed as before forpurification of HaSV virions, the HaSV band with a density of 1.296 g/mlwas again recovered in vast excess to a varying minor amount of the moredense band. No virions of either type were recovered from uninfectedcontrol larvae. Proteins extracted from the more dense particlesappeared identical to those from the less dense particles when examinedby SDS-PAGE and immunoblotting with antibodies specific for the 65 kDacapsid protein of HaSV. Extraction and examination of the RNA genomewith denaturing agarose gel electrophoresis also showed the same 5.5 and2.4 kb bands. When particles from the more dense band were examined byelectron microscopy as before, they appeared to have a larger diameter45 nm but otherwise highly similar to the 38 nm particles.

The molar ratio of the two RNA strands was determined by quantitativedensitometry of fluorograms of the resolved strands. The ratio derivedfrom an average of four measurements of various loadings on denaturinggels proved to be 1.7:1 (5.5 kb strand: 2.4 kb strand) which is somewhatlower than the expected ratio of 2.3:1 for equimolar amounts of eachstrand.

The genome of HaSV has major differences that make it distinct fromthose of the nodaviruses, the only other group of bipartite small RNAviruses pathogenic to animals. Although HaSV shares the characteristicof a bipartite genome with the only animal viruses having such a dividedgenome, the nodaviridae, it differs in virtually every other aspect fromthis group. Both segments of its genome are considerably larger than thecorresponding nodaviral RNAs (Hendry D. A., (1991) Nodaviridae ofInvertebrates. in (ed. E. Kurstak) Viruses of Invertebrates. MarcelDekker, New York, pp. 227-276). However, the division of genetic labouris similar with the larger component carrying the replicase gene and thesmaller one encoding the capsid proteins. Direct comparison of thesequences shows little homology between these viruses, at either RNA orprotein level. The Nodaviruses, have the already mentioned unusual 3′blockage (probably a protein), whereas the HaSV RNAs terminate in adistinctive secondary structure resembling a tRNA.

vii) Bioassays of Virus Isolates on Larvae

The original constructs made to express the capsid proteins (precursorand processed forms) in E. coli for bioassay started at the first AUG(nts 284 to 286). Production of full-length, immuno-reactive proteinfrom these was due to these clones being the 5C sequence version withthe extra C residue. Bioassays of these proteins have been difficult dueto problems with obtaining suitable Heliothis larvae for the tests.

Purified native HaSV was used to conduct bioassays in non-noctuid insectspecies. The native HaSV was orally administered, the larvae scored forsymptoms of infection and growth was measured. Dot blotting for HaSV RNAwas also conducted. Based on these experiments native HaSV does notappear to infect the following larvae. Species Order Family Galleriamellonella Lepidoptera Pyradidae Tineola bissellia Lepidoptera TineidaeEpiphyas postvittana Lepidoptera Tortricidae Lucilia cuprina DipteraCalliphoridae Dacus tyronii Diptera Tephritidae Antitrogus parvulusColeoptera Scarabaediae Lepidiota picticollis Coleoptera ScarabaediaeSericesthis germinata Coleoptera Scarabaediae

The above experiment conducted with the larvae of Spodoptera exigua andS. litura showed that native HaSV infects these species but not to thesame degree as seen in Heliothis armigera.

EXAMPLE 2 Other Virus Isolates

Materials and Methods

A Virus Isolation

Apparently infected (viz diseased) larvae of Helicoverpa sp werecollected in February 1993 at Mullaley (NSW), Narrabri (NSW) andToowoomba (QLD) (Australia). Referring to FIG. 10 the samples in wells2A-2D were from parasitised H. armigera larvae collected from sorghum atMullaley; the sample in 6C was collected from sunflower at Toowoomba;the sample in 7D was collected from cotton at the Narrabri ResearchStation. The latter two larvae may have been either H. armigera or H.punctigera, which are both easily infected with HaSV.

B Virus RNA Extraction

Larvae collected were ground up and RNA extracted. RNA extraction andpurification were as per Example 1.

C Dot-Blot Northern Hybridization

Extracts of viral RNA was analysed by Northern dot-blot hybridisationusing a probe made from cloned HaSV sequences derived from 3′-terminal1000 units of RNA 1 and RNA 2 by random priming in a Boehringer Mannheimkit according to the supplier's instructions were employed. RNA extractswere transferred to Zeta-Probe (BioRad) for probing. Hybridization underhigh stringency washing conditions were as specified by BioRad.Hybridizations were carried out in the following solution:

1 mM EDTA, 500 mM HaH₂PO₄, pH 7.2, 7% SDS, at 65

C in a rotating Hybaid hybridization chamber. After completion ofhybridization and removal of the solution containing the probe, thefilters were washed twice in 1 mM EDTA, 40 mM HaH₂PO₄ pH 7.2, 5% SDS, at65

C (1 h each), followed by 2 washes in 1 mM EDTA, 40 mM HaH₂PO₄, pH 7.21% SDS, at 65

C (1 h each), before autoradiography.

Results

Referring to FIG. 10, samples 9A, 9B, 10A, 10B and 10C contain HaSVinfected positive control lab-raised larvae; 9C-H contain healthy(HaSV-free) negative control lab-raised larvae; All other wells(beginning 1-8) contain extract from field-collected larvae. Numbers2A-D, 6C and 7D gave positive signals indicating that these isolates areeither the same as HaSV or derivatives or variants thereof. Electionmicroscopy employing (−) staining confirmed that the samples which gavepositive signals contained abundant icosohedral virus particles ofapproximately 36mm in size.

The presence of HaSV in larvae which had tested positive in the Northernhybridization dot-blot was confirmed by Western blotting of crudeextracts from such infected larvae, using the polyclonal antibody to theHaSV capsid protein. For routine screening of such extracts in order toidentify fuirther isolates of HaSV or to confirm the presence of thevirus, use of a monoclonal antibody or its equivalent is preferable, inorder to achieve (i) higher sensitivity of detection and (ii) greaterspecificity of detection.

EXAMPLE 3 Identification, Isolation and Characterisation of Insect VirusGenes

Materials and Methods

A Animals and Virus Production.

H armigera larvae were raised as described in Example 1.

B Protein Characterization

Was conducted as described in Example 1.

C Nucleic Acid Characterization

Was conducted as in Example 1.

D Fractionation of Virus RNA

The two viral RNAs were separated by a “freeze and squeeze” method afterresolution on nondenaturing low melting point agarose gels in TAE(Sambrook, et al, 1989). Briefly, agarose slices containing the RNA weremelted at 65° C. in a volume of TAE buffer equal to six times theagarose volume. The solution was allowed to gel on ice before freezingit at −80° C. for 30 minutes. The frozen solution was thawed on ice thencentrifuged at 14,500 g for 10 minutes after which the supernatent waswithdrawn and precipitated by the addition of ethanol.

E In Vitro Translation of HaSV RNA

Was as in Example 1.

F cDNA Synthesis and Cloning of Virus Genome

The virus RNAs were reverse transcribed into cDNA using the SuperscriptRTase (a modified form of the Moloney murine leukaemia virus (MMLV)RTase, produced by Life Technologies Inc). Oligo(dT) was used as aprimer on RNA which had been polyadenylated in vitro. After sizeselection of DNA fragments over 1 kbp in length, the cDNA was thenblunt-end ligated using T4 DNA ligase (Boehringer Mannheim or Promega,under conditions described by the suppliers) into vector pBSSK(−)(Stratagene) which had been cut with EcoRV and dephosphorylated withcalf intestinal alkaline phosphatase (Boehringer Mannheim). E.colistrain JM109 or JPA101 were electroporated with the ligation mixture andwhite colonies selected on colour-indicator plates Sambrook et al, 1989.

For some clones of RNA2 (SEQ ID No: 47), cDNA was synthesised using theRTase of AMV (Promega) and a specific primer complementary to nucleotidesequence 2285-2301 of RNA 2 (SEQ ID No: 47). The same buffer andconditions were used for the Superscript RTase (above). The AMV RTasewas found not to make cDNA form a primer annealing to the terminal 18nucleotide sequence (see below), nor to be able to reach the 5′-end ofthe RNA with the primer here described.

G Sequencing of DNA and RNA

The cDNA clones were separated as single-stranded or double-strandedDNA, using the deaza-dGTP and deaza-dITP nucleotide analogues(Pharmacia) in the deaza T7 sequencing kit as recommended by thissupplier. Synthetic oligonucleotides were used as primers. The 5′terminal sequences of the two RNAs were determined using reversetranscriptase to sequence the RNA template directly, from specificoligonucleotide primers located about 200 nucleotides downstream fromthe termini. Such RNA sequencing was performed using the reversetranscriptase sequencing kit from Promega, under the conditionsdescribed by the manufacturer.

The sequence of the 20 or so nucleotides at the 5′ terminus of each RNAwas checked using direct RNase digestion of 5′-labelled RNA underconditions designed to confer sequence-specificity. Direct RNA sequenceusing RNases was performed with the RNase sequencing kit from USBiochemicals, following the protocols provided by the manufacturer. Thisalso confirmed that the sequence of the most abundant RNA is consistentwith that of the RNA analysed using the specific primer and RTase.

All transcription of plasmids linearized as described were performed asrecommended by the suppliers of SP6 RNA polymerase, in the presence oflmM cap analogue, 0.2mM GTP, and 0.5mM of the other NTPs.

H Subcloning and Expression

PCR Amplification

The polymerase chain reaction (PCR) was used to obtain sequencescovering virus genes in a form suitable for cloning into expressionvectors. The reaction was performed with Taq DNA polymerase (Promega) asdescribed by the supplier, in a rapid cycling thermal sequencermanufactured by Corbett Research (Sydney, Australia). A typical reactioninvolved 1 cycle of 1 min at 90° C., 25 cycles of 95° C. (10 sec), 50°C. (20 sec), 72° C. (1.5 min), followed by one cycle of 72° C. for 5min. Templates were generally cDNA or cDNA clones derived from HaSVRNAs, made as described below. Primers were as described below for therelevant constructs.

Upon termination of the PCR reaction, the product's ends were made bluntby treatment with E.coli DNA polymerase 1 (Klenow fragment) at ambienttemperature for 15 minutes. After heating at 65° C. for 10 minutes, thereaction was cooled on ice and the reaction mix made 1 mM in ATP. Theproduct then 5-phosphorylated using 5 units of T4 polynucleotide kinaseat 37° C. for 30 minutes. After heating at 65° C. for 10 minutes, theproduct was run on a 1% low-melting agarose gel and purified asdescribed for RNA in section E above.

ligations: Vectors and restriction fragments cut with the enzymesdescribed were run on 1% low-melting-point agarose gels and excised asslices. These slices were then melted at 65° C. for 5 minutes, beforecooling to 37° C. Fragment and vectors were then ligated in 10 ul totalvolume at 14° C. overnight using T4DNA ligase (BRL, Boehringer Mannheimor Promega), in the buffers supplied by the manufacturers.

expression: Expression plasmids containing viral genes (e.g. for thecapsid protein) were transformed into E. coli strain BL21 (DE3) or HMS174 (DE3) (supplied by Novagen). After growth as specified by thesupplier, protein expression was induced by the addition of isopropylb-D-thiogalactopyranoside (IPTG), at 0.4 nM to the growing culture for aperiod of 3 h. Expressed proteins were analysed by SDS-polyacrylamidegel electrophoresis of bacterial extracts (Laemmli, 1970).

Results

i) Mapping cDNA Clones of HaSV

The template for cDNA synthesis was virus RNA which had beenpolyadenylated in vitro. Oligo(dT) was used as a primer for theSuperscript reverse transcriptase (RTase; a modified form of the Moloneymurine leukaemia virus (MMLV) RTase, produced by Life Technologies Inc).The cDNA was cloned into vector pBSSK(−) as described earlier. Thelarger clones were selected for further analysis by restriction mappingand Northern hybridization. All the probes tested hybridized either toRNA 1 or to RNA 2, suggesting that there are no regions of extensivesequence homology between the two RNA's. Furthermore, screening of anumber of other clones excluded the theoretical possibility that eitherRNA band may actually contain more than one species.

ii) RNA 1 (SEQ ID No: 39) Clones

Three large RNA 1 (SEQ ID No: 39) clones (B11U, B11O and B35) obtainedfor the first round of clones were further analysed by restrictionmapping and shown to form an overlap spanning over 3 kbp (this was laterconfirmed by sequencing). The second round of cloning then yielded E3 of5.3 kbp, representing 99.7% of RNA 1 (SEQ ID No: 39). A completerestriction map of clone E3 showed it to align with that previouslydetermined for three overlapping clones. On the basis of this alignment,the 5′ end of the insert in B11U was placed about 300 nucleotidesdownstream from the 5′ end of the RNA.

Once clones covering a contiguous block had been identified, theorientation 3relative to the RNA was determined.

iii) RNA 2 (SEQ ID No: 47) Clones

Three significant cDNA clones were isolated for RNA 2 (SEQ ID No: 47)(FIG. 2). One, hr236, contains about 88% of RNA 2 (SEQ ID No: 47) (2470bp total length), and runs from the 3′ end to 240 bp from the 5′ end.The other clones, hr247 and hr 249 are 3′ coterminal subgenomicfragments of 1520 bp and 760 bp, respectively. Orientation of clonehr236 was determined by strand specific hybridization. While a muchstronger signal was seen with a probe for one orientation, the probespecific for the other orientation also yielded a signal, indicatingthat there are extensive regions of reverse complementarity within thepositive strand sequence. Such sequences are likely to form extensiveshort and long-range secondary structure.

The clones contain the 3′ sequence of HaSV RNA 2 (SEQ ID No: 47) as theyall have the same 3′ sequence adjacent to the poly (A) stretch added invitro before cDNA priming. The remaining 5′ sequence of RNA 2 has beenobtained by direct RNA sequencing using two reverse transcriptases asdescribed above.

iv) Sequencing of Virus Genome

The clones mapped in section (i) were selected for further analysis bysequencing.

The cDNA clones were completely sequenced as single-stranded DNA in bothorientations, using the deaza-dGTP and deaza-dITP nucleotide analogues(Pharmacia) and synthetic oligonucleotides as primers.

v) Sequence of Genome Component 1 (SEQ ID No: 39) (See FIG. 1)

The 5310 nucleotides of RNA 1 (SEQ ID No: 39) encode a protein ofmolecular weight 187,000 which is regarded as the RNA-dependent RNApolymerase (replicase) in view of its amino acid sequence similarity incertain limited regions to replicases of other RNA viruses. The apparentmolecular weight of this protein upon in vitro translation of virus RNAand SDS-PAGE is 195,000.

Sequence analysis of RNA 1 (SEQ ID No: 39) was concentrated on clone E3which extends from the 3′ end of RNA 1 to 18 nucleotides form the 5′ end(FIG. 1). The complete sequence has been confirmed by sequencing in bothdirections. An ORF of 1750 amino acids and spanning virtually thecomplete RNA (5310 nucleotides in length) has been detected. This ORFbegins with the first AUG on the sequence at position 34 and terminatesat nucleotide 5290 and is thought to encode the RNA-dependent RNApolymerase (replicase)(referred to as P187 (SEQ ID No: 40) in FIG. 1)required for virus replication, since it contains the Gly-Asp-Aspconserved triplet and surrounding sequences identified in these enzymes,which are usually large (over 100 kDa), in addition to further homologywith the polymerase encoded by tobacco mosaic virus and otherplus-stranded RNA viruses.

Referring to FIG. 1 the sequence is presented as the upper strand of thecDNA sequence. This strand is therefore in the same sense as the viral(positive-sense) RNA. The sequence of the protein encoded by the majoropen reading frame, encoding the putative RNA-dependent RNA replicase,is shown, as are those of the small open reading frames at the 3′ end,corresponding to the proteins P11a (SEQ ID No: 42), P11b (SEQ ID No: 44)and P14 (SEQ ID No: 46).

Clone E3 was inserted downstream of the SP6 promoter for in vitrotranscription. As mentioned above, the transcript of this clone can betranslated in the wheat germ system to yield the 195 kDa proteinobserved upon translation of fractionated RNA 1 (SEQ ID No: 39) from thevirus. The latter yields more lower molecular weight products,presumably due to being contaminated with nicked and degraded RNA. Theproducts derived from the in vitro transcript can therefore be regardedas defining the coding capacity of the complete RNA 1 (SEQ ID No: 39) ofHaSV.

vi) Sequence of Genome Component 2 (See FIG. 2)

The 2470 nucleotides encode a protein of molecular weight 71,000 whichcontains the peptide sequences corresponding to those determined fromthe two virus capsid proteins. This protein is therefore the precursorof these capsid proteins. The protein is a major product of in vitrotranslation of this RNA obtained either from virus particles or by invitro transcription of a full-length cDNA clone; in addition, anothermajor translation product of apparent molecular weight 24,000 isobtained. This protein is derived from a molecular weight 17,000 readingframe overlappling the slab of the capsid protein gene.

Clones hr236 and hr247 were completely sequenced as the first step inRNA 2 sequencing. These sequences were then extensively compared to thatobtained by direct RNA sequencing using AMV reverse transcriptase.

Comparison of the cloned sequence with that by direct RNA sequencingshowed both clones lacked 50 nucleotide present in the RNA (at aroundnucleotide 1500). The sequence of this stretch was obtained by directRNA sequencing using the AMV RTase. The MMLV “Superscript” RTase, whichwas used to make all the cDNA clones, was found to simply by-pass thisregion in sequencing reactions. These 50 nucleotides contain a verystable GC-rich hairpin flanked by a 6 bp direct repeat, and the MMLVRTase skips from the first repeat to the second.

The sequence of RNA 2 (SEQ ID No: 47) was then completed using plasmidspSR2A and pSR2P70 constructed as described below. The plasmids contain asegment of cDNA derived for the AMV RTase, as well as the sequencecorresponding to the 5′ 240 nucleotides of RNA 2 (SEQ ID No: 47) whichare not present on phr236 (FIG. 2). The sequence of RNA in FIG. 2 ispresented as the upper strand of the cDNA sequence. This strand istherefore in the same sense as the viral (positive-sense) RNA. Thesequences of the proteins encoded by the major open reading frames,encoding the capsid protein precursor P71 (SEQ ID No: 50), and P17 (SEQID No: 48).

The sequence of RNA 2 (SEQ ID No: 47) encodes a major ORF running from amethionine initiation codon at nucleotides 366 to 368 to a terminationcodon at nucleotides 2307 to 2309. This protein encoded by this ORF hasa theoretical molecular weight of 71,000 (SEQ ID No: 50). Thisinitiation codon is in a good context (AGGatgG), suggesting that it willbe well recognized by scanning ribosomes. The size of the product isclose to that of the residual putative precursor protein identified inpurified virus, and to the size of the in vitro translation productobtained from RNA 2 (SEQ ID No: 47).

The approach adopted to identify the gene encoding the capsid proteinwas to obtain amino acid sequence information from the two abundantcapsid proteins and then locate these on the protein encoded by thesequence of the virus RNA's. CNBr cleaved products of the capsid proteinwere therefore sequenced. These fragments gave a clear and unambiguoussequence shown in Example 1. These sequences determined were thenlocated on the large ORF of RNA 2 (SEQ ID No: 47). (FIG. 2)

In the case of the small capsid protein, the clear and unambiguoussequence, obtained is located near the carboxy terminus of the major ORFon RNA 2 (SEQ ID No: 47). Starting at the point corresponding to theamino-terminal residue of the sequence determined for the 6 kDa protein,and continuing to the carboxy-terminus of the complete reading frame,the protein encoded by the sequence 7.2 kDa and has a hydrophobicN-terminal region and an arginine rich (basic) C-terminal region. It isan extremely basic protein with a pI of 12.6.

The two abundant capsid proteins are derived from a single precursor,which is processed at a specific site. This is presumably immediatelyamino-terminal to the sequence FAAAVS . . . (SEQ ID No: 25)

RNA 2 (SEQ ID No: 47) appears to be a bicistronic mRNA (see FIGS. 2 and5). The first methionine codon is encoded on the sequence of RNA atnucleotides 283 to 285. This ATG is in a poor context (TTTatgA), makingit a weaker initiation codon. It initiates a reading frame of 157 aminoacids, encoding a protein of molecular weight 17,000 (SEQ ID No: 48).(The second AUG [nts 366 to 368] initiates the 71 kDa (SEQ ID No: 50)precursor of the capsid protein). Since the first AUG is in a poorcontext, abundant expression of the capsid precursor would be expected.In fact, in vitro translation of a full length RNA 2 (SEQ ID No: 47)transcribed from a reconstructed cDNA clone yields two major proteinproducts of relative mobility 71,000 (SEQ ID No: 50) and 24,000, similarto those already observed upon translation of viral RNA 2 (SEQ ID No:47). The protein of Mr 24,000 appears to correspond to the 157 aminoacid protein, despite the significant anomaly in apparent size. The24,000 Mr product was also observed upon translation of an in vitrotranscript covering only nucleotides 220 to 1200 of RNA 2 (SEQ ID No:47). This region contains no open reading frame other than those alreadymentioned and cannot encode a protein longer than 157 amino acids.

The protein of Mr 24,000 seen upon in vitro translation appears tocorrespond to P17 (SEQ ID No: 48), with the anomaly in apparent sizeprobably being due to the high content of proline (P), glutamate (E),serine (S) and threonine (T). These amino acids cause the protein runmore slowly on a gel thereby giving it an apparent size of Mr 24,000.

The Mr 24,000 protein (hereinafter referred to as P17 (SEQ ID No: 48))may have a function in modifying or manipulating the growthcharacteristics or cell cycle of HaSV-infected cells. Although a proteinof 16 kDa (identified in Example 1) is found in small amounts in thecapsid, it does not react with antiserum against the virus particlesthis is unlikely to correspond to P17 (SEQ ID No: 48), since apreparation of the latter proteins migrates with a molecular weight of24,000 on SDS gels.

Sequence analysis of the Region from nucleotide 500 to 600 of RNA 2 (SEQID No: 47) showed that it has the sequence shown in FIG. 2, as do theplasmids pSR2A, pSR2P70, pSR2B and pSXR2P70. However, plasmids pT7T72P65and pT7T2P70 have an extra C residue at nucleotide 570. The RNA sequencefrom which they are derived from is shown in FIG. 2 (the “5C” version).In this sequence the first ATG (nucleotides 283 to 285) is in the samereading frame as most of the capsid protein gene. The resultant fusionprotein is called “P70” (SEQ ID No: 52) and itscarboxyterminal-truncated version (a variant of the native P64) is“P65”. In view of these clones it was considered important to resolvewhether any virus RNA carrying the extra C residue was present in theviral RNA population first isolated for investigation.

Direct sequencing of the virus RNA using reverse transcriptase confirmedthat the 4C version lacking the extra residue was the abundant form ofthe RNA. In order to exclude the possibility of a small amount of theRNA having the extra residue, a sensitive PCR assay was designed. Thisshowed that the extra C residue was not present on any RNA in the viralpopulation, and had been introduced into some clones as a PCR artefact.These clones were however retained and used in bacterial expressionexperiments (below) because of the high level expression obtained of theP65 and P70 (SEQ ID No: 52) fusion proteins.

vii) Comparison with the Sequence of the Nudaurelia w Capsid Gene

The sequence of most of the RNA2 of the Nudaurelia w virus has recentlybeen published by Agrawal D. K. and Johnson J. E. (Virology 190 806-814,1992). From the published sequence it has been determined that thissequence shows 63% homology to that of HaSV RNA2 (SEQ ID No: 47) at thenucleotide level and 66% at the overall amino acid level. A detailedcomparison of the capsid proteins of these two viruses shows theamino-terminal 45 residues to be variable, the next 220 residues to behighly conserved, the next 180 residues to be variable and thec-terminal 200 residues covering the small protein P7 to be highlyconserved. A more detailed comparison is discussed below.

The published report did not find a complete reading frame correspondingto the 157 amino acid protein (P17 (SEQ ID No: 48)) gene reported above.The AUG is however present, as is a reading frame—starting upstream ofthe start of the capsid gene—showing considerable amino acid homology toP17 (SEQ ID No: 48) of HaSV. In vitro translation of purifiedNudazirelia w virus RNA 2 and a re-examination of the nucleotidesequencing data for this RNA may help to resolve the question of whetherthe Nudaurelia w virus also encodes a protein homologous to the HaSVP17.

More interestingly, antisera against these two viruses, which aresimilar at a nucleotide sequence level, do not show anycross-reactivity.

viii) Construction of Full-Length Clones

RNA 1 (SEQ ID No: 39)

cDNA clone E3, described above contains all but the 5′-18 nucleotides ofRNA 1 (SEQ ID No: 39) and included the complete ORF present on thesequence. The first full-length clone of RNA 1 (SEQ ID No: 39) istherefore based on E3. The 4.9 kbp XbaI-ClaI fragment from clone E3 wasrecloned into pBSKS(−) (Stratagene) cut with XbaI and Clal, givingpBSKSE3.

The full-length clone of RNA 1 (SEQ ID No: 39) was completed using PCR.The primer defining the 5′ end of the RNA carried an EcoRI site, thepromoter for the SP6 RNA polymerase and a sequence corresponding to the5′ 17 nucleotides of RNA 1, as shown in FIG. 1. The sequence of thisprimer was:

HvR1SP5p:

5′ -GGGGGGAATTCATTTAGGTGACACTATAGTTCTGCCTCCCCGGAC (SEQ ID No: 11) (The Gwhich initiates transcription is underlined)

Using an oligonucleotide complementary to nucleotides 1192-1212, a PCRproduct of 1240 bp was efficiently made. The template was cDNAsynthesised using the MMLV RTase and the same oligonucleotidecomplementary to nucleotides 1192-1212 was the primer. Upon terminationof the PCR reaction, the product's ends were made blunt and then5′-phophorylated as described below. The purified PCR fragment was thencleaved with restriction endonuclease XbaI and the 450 bp subfragmentcorresponding to the 5′ end of RNA 1 (SEQ ID No: 39) cloned into theplasmid pBSSK(−)(Stragene) cut with EcoRV and XbaI, to give pBSR15.

To assemble the full-length of RNA 1 (SEQ ID No: 39), pBSKSE3 (above)was cut with XbaI and ScaI giving fragments of 1.2 kbp and 6.8 kbp.pBSR15 was cut with the same enzymes, giving fragments of 2 and 1.8 kbp.Ligation of the 6.8 kbp fragment for pBSKSE3 and the 1.8 kbp fragmentfor mpBSR15 yielded pSR1(E3)A. Upon linearization at ClaI and in vitrotranscription with the SP6 RNA polymerase, and RNA corresponding to RNA1 (SEQ ID No: 39), and terminating in a poly(A) stretch of about 50nucleotides, is obtained.

Since the natural RNA 1 (SEQ ID No: 39) does not have a poly (A) tail,an alternative plasmid was constructed which carries a BamHI restrictionsite immediately downstream of the 3′ end of RNA 1 (SEQ ID No: 39).Again this terminal fragment was made using PCR as above. The sequenceof the primer was as follows:

HvR13p: 5′-GGGGGGATCCTGGTATCCCAGGGGCGC (SEQ ID No: 12) (the nucleotidecomplementary to that which was determined as the 3′ one, based on itsadjacency to the poly(A) stretch, is underlined; RNA terminating at theBamHI site will have the sequence GCGCCCCCUGGGAUACCaggauc (SEQ ID No:26)).

The template was clone E3 and an oligonucleotide corresponding tonucleotides 4084-4100 was the other primer. The 1220 bp product wasblunt-ended, kinased and gel-purified as described above, beforecleavage with HindIII. The resulting 420 bp subfragment corresponding tothe 3′ end of RNA 1 (SEQ ID No: 39) cloned into plasmid pSRl(E3)A cutwith Clal, end-filled with Kienow and then cut with HindIII. Theresulting plasmid is pSR1(E3)B. Upon linearization at BamHI and in vitrotranscription with the SP6 RNA polymerase, and RNA corresponding to RNA1 (SEQ ID No: 39), and terminating as described immediately above isobtained.

ix) RNA 2 (SEQ ID No: 47)

In constructing the full-length cDNA clone to enable in vitrotranscription of this RNA hr236 described above was used as a basis. Twoseparate PCR products, one corresponding to the 5′ portion of RNA 2 (SEQID No: 47), which is missing from this clone altogether, and anothercovering the region where clone hr236 lacks the hairpin-forming sequencedescribed above, were required.

The primer defining the 5′ end of the RNA carried a HindIII site and asequence corresponding to the 5′ 18 nucleotides of RNA 2 (SEQ ID No:47), as shown in FIG. 2. The sequence of this primer was: (SEQ ID No:13) Hr2cdna5: 5′-CCGGAAGCTTGTTTTTCTTTCTTTACCA(The nucleotide underlined corresponds to that identified as the firstnucleotide of RNA 2. (SEQ ID No: 47))

Using an oligonucleotide complementary to nucleotides 1653-1669, a PCRproduct of 1.67 kbp was made. The template was cDNA synthesised usingthe MMLV RTase and an oligonucleotide complementary to the 18nucleotides at the 3′ end of RNA 2 (SEQ ID No: 47) as the primer. Upontermination of the PCR reaction, the product was blunt-ended, kinasedand gel-purified as described above, before cleavage with PstI. Theresulting 1.3 kbp subfragment corresponding to the 5′ half of RNA 2 (SEQID No: 47) was cloned into plasmid pBSSK(−) (Stragene) cut with EcoRVand PstI, giving plasmid pBSR25p. In order to place this subfragmentcorresponding to the 5′ half of RNA 2 (SEQ ID No: 47) downstream of theSP6 promoter for in vitro transcription, a 1.3 kbp HindIII—BamHIfragment was excised from pBSR25p and ligated into HindIII—BamHI cutpGEM-1 (Promega), giving plasmid pSR25.

The second PCR product, covering the region where clone hr236 lacks thehairpin-forming sequence described above, was synthesised using asprimers oligonucleotides corresponding to nucleotide sequence 873 to 889of RNA 2 (SEQ ID No: 47) and to the complement of nucleotide sequence2290-2309. Upon termination of the PCR reaction, the product wasblunt-ended, kinased and gel-purified as described above, beforecleavage with AatII. The resulting 1.1 kbp subfragment covering therequired region was cloned into plasmid phr236 cut with HindIII,end-filled with Klenow and cut with AatII, giving plasmid phr236P70.

The two segments were joined covering the first 230 nucleotides of RNA 2(SEQ ID No: 47) together. Plasmid phr236P70 was cut at the SacI site inthe vector adjacent to the 5′ end of the insert and this madeblunt-ended using Klenow in the absence of dNTPs. Afterheat-inactivation of the Klenow, the plasmid was cut with EcoRI,yielding fragments of 4.5 kbp and 380 bp. Plasmid pSR25 was cut withNheI, blunt-ended by end-filling with Klenow and cut with EcoRI,yielding fragments of 2.8 kbp, 900 bp and 750 bp. The 4.5 kbp fragmentof phr236P70 and the 900 bp fragment of pSR25 were ligated to givepSR2P70. This clone covers all of RNA 2 (SEQ ID No: 47) except for the3′ 169 nucleotides.

To complete the full-length clone of RNA 2 (SEQ ID No: 47), it wasnecessary to insert a fragment covering the 3′ end. As with RNA 1 (SEQID No: 39), two versions were made. One, called pSR2A, used the 3′ endas present in phr236, together with the poly(A) tail present in thisversion. The other pSR2B, used a PCR fragment carrying a BamHI siteimmediately downstream of the 3′ nucleotide, as in pSR1(E3)B above. Toconstruct pSR2A, a 350 bp NotI-ClaI fragment was excised from phr236 andcloned into pSR2P70 cut with the same endonucleases. Linearization atthe unique ClaI site allows in vitro transcription of the complete RNA 2(SEQ ID No: 47) and a poly(A) tail of about 50 nucleotides in length.

To make pSR2B, an appropriate PCR product was made using as primers anoligonucleotide corresponding to nucleotide sequence 1178 to 1194 and tothe 3′ terminal 18 nucleotides of RNA 2 (SEQ ID No: 47). The latterprimer carried a BamHII site attached, giving it the sequence: (SEQ IDNo: 14) HvR23p: 5′-GGGGGATCCGATGGTATCCCGAGGGACGC

The template used was a plasmid phr236. Upon termination of the PCRreaction, the product was blunt-ended, kinased and gel-purified asdescribed above, before cleavage with Noti. The resulting 400 bpsubfragment covering the required region was cloned into plasmid pSR2P70cut with Clal, end-filled with Klenow and cut with NotI, giving plasmidpSRP2B. Linearization at the unique BamHI site allows in vitrotranscription of the complete RNA 2 (SEQ ID No: 47), terminating withthe sequence ACCaggatc.

x) Construction of pSXR2P70

This plasmid was made to determine where p24 starts. A 2.1 kbpXhoI-BamHI fragment was cut from clone pSR2P70 and ligated into thevector pGEM-1 (Promega) which had been cut with SalI and BamHI. In vitrotranscription of the resulting plasmid after linearization at the uniqueBamHI site yielded an RNA covering about 70 nucleotides upstream of thefirst ATG at nucleotides 283 to 286, plus a short sequence derived fromthe vector.

In vitro translation of the RNA from pSXR2P70 yielded both proteins (P70(SEQ ID No: 52)+P24).

xi) Description of Virus-Induced Pathology

The virus induces a rapid anti-feeding effect in Helicoverpa larvae asdetermined by experiments with larvae the results of which are shown inFIG. 3. FIG. 3 shows: A. neonate larvae (less than 24 h old) were fedthe designated concentrations of isolated virus (in particles per ml [ofdiet] added to solid diet). They were weighed on following days and themean of a statistically significant number (24) of larvae shown. Wherenecessary, mortality was recorded for the higher concentrations. Thevertical axis shows the fold-increase in weight from the hatching weightof 0.1 mg per larvae. This scale therefore also corresponds to weight inunits of 0.1 mg (ie 300 is equivalent to 30 mg). B. As for A, but thelarvae were 5 days old at the start of the virus feeding. The verticalscale is in mg weight.

No weight gain at all was detectable with neonates which had been fedthe doses of virus over 10⁸ particles per ml (virus added to diet). Inaddition, 100% mortality was evident after four days at the highestdoses. Virus doses as low as 10⁶ particles per ml (virus added to diet)still cause significant stunting. The five day old larvae showed acessation of feeding after 48 hours and significant stunting at 4 dpi,but no mortality at comparable virus doses (FIG. 3). Neonates aretherefore very sensitive indeed to this virus. Virus particlesaccumulate specifically in the midgut. This potent anti-feeding effectmay be due to the capsid protein or another protein encoded by thevirus, or to the effect of any combination of such proteins.

xii) Expression of Virus-Encoded Proteins in Bacteria.

The vectors

The expression system used initially was derived from the pET-11 system(Novagen). Trimmed down versions of pET-11b and c were constructed andused to compare expression of the capsid proteins. However, due todifficulties experienced with this system substantial modification ofthe original vectors was carried out in order to achieve much higheryields. These results are described in xiii-b) below.

The initial trimmed-down vectors discussed above were made as follows:pGEM-2 (Promega) which carries T7 promoter adjacent to a poly-linkersequence, but has no sequences corresponding to the lac operon, was cutat the unique XbaI (34) and Scal (1651) sites, giving fragments of 1.61and 1.25 kbp. The plasmids pET-11b and c were cut with the same enzymes,giving fragments of 4.77 and 0.91 kbp. The 1.61 kbp fragment of pGEM-2,carrying the c-terminal portion of the ampicillin-resistance gene, theorigin of replication and the T7 promoter, was then ligated to the 0.91kbp fragment of the pET vector, which carries a sequence covering theShine-Dalgamo sequence, the ATG (in a NdeI site), the terminator for theT7 polymerase and the N-terminal portion of the ampicillin-resistancegene. The resulting plasmids of approximately 2.53 kbp, called pT7T2-band c, therefore carry a complete T7 transcription unit, which may beused as an expression system in a manner similar to the original pET-11plasmids, but are repressor-neutral within the cell; they neithertitrate away repressor by carrying a binding site, nor do they carry thegene producing the repressor. They were found to grow very well inE.coli strains JM109 and BL21 (DE3), and to be very efficient expressionvectors. The repressor present in the cells was found to be sufficientto keep the genomic T7 polymerase gene uninduced and therefore theforeign gene unexpressed in the absence of IPTG.

xiii-a) Construction of Plasmids for Expression of Capsid Proteins

In this section, all proteins expressed from segments of HaSV RNA 2 (SEQID No: 47) are referred to by the size of their gene, as defmed in FIG.4 and in section vi) of this example. The following plasmids wereconstructed by PCR, using the abovementioned full-length clone of RNA 2(SEQ ID No: 47), plasmid pSR2A as the template, except where mentionedotherwise.

Groups of plasmids expressed protein starting at each of the first threemethionine initiation codons found on the sequence of HaSV RNA 2 (SEQ IDNo: 47). For those proteins initiating at the first methionineinitiation codon found on the sequence of HaSV RNA 2 (SEQ ID No: 47)(which initiates the P17 (SEQ ID No: 48) gene; oligonucleotide primerHVPET65N (SEQ ID No: 15)), an extra group of plasmids was made by PCRusing as a template the version of the RNA 2 sequence carrying an extraC residue inserted at residue 570 (SEQ ID No: 51) (as depicted in FIG.2). Expression constructs initiating at the third methionine initiationcodon found on the sequence of HaSV RNA 2 (which is located within theP17 gene; oligonucleotide primer HVPET63N (SEQ ID No: 16)) were made byPCR using as a template only the version of the RNA 2 sequence carryingan extra C residue inserted at residue 570 (SEQ ID No: 51). For theselatter expression constructs, as well as those designed to initiateexpression from the second methionine initiation codon found on thesequence of HaSV RNA 2 (SEQ ID No: 47) (which initiates the P71 gene;oligonucleotide primer HVPET64N (SEQ ID No: 17)), two versions wereconstructed.

One version terminated at a point corresponding to the c-terminus of theprocessed (P64) form of the capsid protein and was made usingoligonucleotide primer HVP65C (SEQ ID No: 19). The other versionterminated at a point corresponding to the c-terminus of the precursor(P71 (SEQ ID No: 50)) form of the capsid protein and was made usingoligonucleotide primer HVP6C2 (SEQ ID No: 20).

The sequence encoding P64 (or the precursor, P71 (SEQ ID No: 50)) wassynthesised in two segments using PCR. The amino-terminal half of thegene was obtained using as primers oligonucleotides incorporating one ofthe three ATG possible initiation codons for the ORF, in addition to anoligonucleotide with the sequence TCAGCAGGTGGCATAGG (SEQ ID No: 27);complementary to nucleotides 1653 to 1669 of the sequence shown in FIG.2. The forward primers were as follows: HVPET65N: (SEQ ID No: 15)AAATAATTTTGTTTACTTTAGAAGGAGATATACATATGAGCGAGCGAGCA CAC

(the underlined sequence corresponds to nucleotides 283 to 296 of thesequence shown in FIG. 2) HVPET63N (SEQ ID No: 16) AAATAATTTTGTTTAACCTTA AGAAGGAGAT C TACATATGCTGGAGTGGC GTCAC

(the underlined sequence corresponds to nucleotides 373 to 390 of thesequence shown in FIG. 2; the AflII (CTTAAG) and BglII (AGATCT) sitesintroduced into the sequence by single nucleotide changes (shown initalics) in the oligonucleotide are shown in bold). (SEQ ID No: 17)HVPET64N GGAGATCTACATATGGGAGATGCTGGAGTG(the underlined sequence corresponds to nucleotides 366 to 383 of thesequence shown in FIG. 2; the BglII site introduced into the sequence bya single nucleotide change in the oligonucleotide is shown in bold).

The PCR products obtained from each combination of one of these primerswith the abovementioned one were treated with the Klenow fragment ofE.coli DNA polymerase, and then with T4 polynucleotide kinase in thepresence of 1 mM ATP, before purification by agarose gel electrophoresisas described above. Each product was then cleaved with AatII to yieldfragments of 0.95 and 0.4 kbp, and each resulting fragment of about 0.95kbp cloned intro vector pGEM-2 (Promega) cut with Hincll and AatII,giving plasmids pGEMP63N (in which the insert commenced witholigonucleotide HVPET63N (SEQ ID No: 16)), pGEMP64N (in which the insertcommenced with oligonucleotide HVPET64N (SEQ ID No: 17)) and pGemP65N(in which the insert commenced with oligonucleotide HVPET65N (SEQ ID No:15)). The fragment covering portion of the HaSV capsid gene was thenexcised with enzymes AatII and XbaI.

Two versions of plasmid pGemP65N were made, using different templates asdescribed above. pGemP65N was derived from the sequence of the viralRNA, as in plasmid pSF2A; plasmid pGemP65Nc was derived from thesequence carrying an extra C residue, as shown in FIG. 2 (see “5Cversion”).

In parallel, the carboxy-terminal halves of the major capsid proteinvariant, whether terminating as for P64 or for P71 (SEQ ID No: 50), werealso produced using PCR. An oligonucleotide primer, HVRNA2F3, with thesequence GTAGCGAACGTCGAGAA (SEQ ID No: 18) (corresponding to nucleotides873 to 889 of the sequence shown in FIG. 2) was used in conjunction witheach of the two primers following: (SEQ ID No: 19) HVP65CGGGGGATCCTCAGTTGTCAGTGGCGGGGTAG

(the underlined sequence is complementary to nucleotides 2072 to 2091 ofthe sequence shown in FIG. 2). (SEQ ID No: 20) HVP6C2GGGGATCCCTAATTGGCACGAGCGGCGC(the underlined sequence is complementary to nucleotides 2290 to 2309 ofthe sequence shown in FIG. 2).

The PCR products obtained from each combination of one of these primerswith the above mentioned one (HvRNA2F3 (SEQ ID No: 18)) were treatedwith the Klenow fragment of E.coli DNA polymerase, and then with T4polynucleotide kinase in the presence of 1 mM ATP, before purificationby agarose gel electrophoresis as described above. Each product was thencleaved with AatII to yield fragments of 0.9 kbp (in the case of HVP65C(SEQ ID No: 19)) or 1.1 kbp (in the case of HVP6C2 (SEQ ID No: 20)) and0.4 kbp, and each resulting fragment of about 0.9 or 1.1 kbp cloned intoplasmid phr236 cut with HindIII, treated with Klenow and AatII, givingplasmids phr236P65C and phr236P70 (which has already been describedabove), respectively. The fragment covering the c-terminus of the capsidprotein gene was then excised with enzymes AatII and BamHI.

To assemble plasmids for expression in suitable strains of E. coli, theexcised XbaI-AatII fragments of 0.95 kbp covering the amino-terminalhalf of the gene and the excised AatII—BamHI fragments of 0.9 or 1.1 kbpcovering the carboxy-terminal half of the gene were simultaneouslyligated into the vector pT7T2 cut with XbaI and BamHI. Initialtransformation was of E. coli strain JM109. Recombinant plasmidscarrying the correct insert were then transformed into strain BL21 (DE3)for expression as described above.

The plasmid obtained by ligating the aminoterminal fragment commencingwith oligonucleotide primer HVPET63N (SEQ ID No: 16) to the c-terminalfragment ending at oligonucleotide primer HVP65C (SEQ ID No: 19) in theepxression vector pT7T2b was called pP65G.

In the case of plasmid pP64N, containing an insert from HVPET64N (SEQ IDNo: 17) to HVP65C (SEQ ID No: 19), the fragment covering theamino-terminal half of the oligonucleotide was excised by BglII and ScaIfrom the plasmid pGemP64N and the fragment covering the remainder of thegene was excised with ScaI and EcoRI from plasmid pT7T2-P65. These twofragments were then ligated simultaneously into pP65G which had been cutwith BglII sand EcoRI.

The resulting construct carrying the complete P71 (SEQ ID No: 50)precursor gene was called pT7T2-P71 and that carrying the P64 form ofthe gen was called pT7T2-P64. In the case of plasmids derived frompGemP65N and pGemP65Nc, carrying inserts commencing as defmed by primerHVPET65N, the expression plasmid derived from pGemP65N which is based onPCR products made using as the template the sequence of the viral RNA,as in plasmid pSR2A, was called pTP17; a truncated form of this plasmid,which expresses P17 (SEQ ID No: 48), was made by cutting at the uniqueBglII and BamHI sites, removing the intervening fragment (whichcorresponds to the c-terminal part of the insert) and religating thecompatible cohesive ends, to give pTP17delBB. The expression plasmidsderived from plasmid pGemP65Nc (which was derived from the sequencecarrying an extra C residue, were called pT7T2-P65 (carrying an insertterminating at the primer HVP65C (SEQ ID No: 19)) and pT7T2-P70(carrying an insert terminating at the primer HVP6C2 (SEQ ID No: 20)).

Expression of P6

Two forms of this protein, which arises through processing of the largecapsid protein variant precursor P70 (SEQ ID No: 52) and therefore lacksits own initiation codon, were made. One form (protein MA) replaced thephenylalanine at the start of this protein with methionine, giving itthe amino-terminal sequence MAA . . . ; the other carries an additionalmethionine residue, giving it the amino-terminal sequence MFAA . . . Theoligonucleotides used for PCR-amplified products covering the p6 codingsequence carried a NdeI site (bold) at the ATG codon, for directligation into the pET-11 vectors. The primers used were: (SEQ ID No: 21)HVP6MA: AATTACATATGGCGGCCGCCGTTTCTGCC (SEQ ID No: 22) HVP6MF:AATTACATATGTTCGCGGCCGCCGTTTCT

Each of these primers was used in conjunction with primer HVP6C2 (SEQ IDNo: 20) to generate a PCR product of 0.2 kbp. These products wereblunt-end ligated into vector pBSSK(−) which had been cut with EcoRV anddephosphorylated. The insert corresponding to the p6 gene was excisedwith Ndel and BamHI (using the BamHI site in the primer HVP6C2 (SEQ IDNo: 20)) and ligated into the expression vector pET-11b, which had beencut with the same enzymes. For expression at higher levels, the insertwas transferred to PT7T2 as a XbaI—BamHI fragment, yielding plasmidspTP6MA and pTP6MF.

IPTG induction of bacteria containing plasmids pTP6MA or pTP6MF wereused produce p6 for bioassay.

xiii-b) Expression of Viral Genes in E. coli and Bioassay in LarvaeExpression of P64

IPTG induction of bacteria containing plasmid pT7T2-P65, which containsan insert running from the location of primer HVPET65N (SEQ ID No: 15)to that of primer HVP65C (SEQ ID No: 19), yielded a protein of molecularweight 68 000. This was 3 000 molecular weight greater than the size ofthe authentic coat protein, as expected. Expression of pP65G, whichcontains an insert running from HVPET63N (SEQ ID No: 16) to HVP65C (SEQID No: 19), yielded a protein of 65 000 molecular weight.

The authentic capsid protein (P64) was expressed poorly from plasmidpT7T2-P64. Recloning this insert as a NdeI-BamHI fragment back into theother form of the vector (PT7T2b) did not alter this.

Expression of P70

IPTG induction of bacteria containing plasmid pT7T2-P70, which containsan insert running from the location of primer HVPET65N (SEQ ID No: 15)to that of primer HVP6C2 (SEQ ID No: 20), yielded a protein of molecularweight 73 000. This was 3 000 molecular weight larger than the size ofthe precursor of the coat protein, as expected.

The authentic capsid protein precursor (P71 (SEQ ID No: 50)) wasexpressed poorly from plasmid pT7T2-P71. Recloning this insert as aNdeI-BamHI fragment back into the other form of the vector (pT7T2b) didnot alter this.

Due to the observation mentioned in vi) above, plasmids designed toexpress all forms of the capsid proteins from several possible ATG's atthe start of the open reading frame were constructed.

It was found that both authentic P64 and P71 (SEQ ID No: 50) wereexpressed poorly in bacteria. In contrast, P17 (SEQ ID No: 48) and theforms of the capsid protein commencing at the P17 ATG were expressedvery well. The extra C residue present in the latter two constructsresulted in a fusion protein being made from these expression plasmid.The sequence of the fusion proteins can be derived from FIG. 2 byincluding an extra C at position 570. The fusion caused the first 67residues of the HaSV capsid protein to be replaced by the first 95residues of P17 (SEQ ID No: 48). Good expression of the large capsidprecursor and protein was achieved, but the size of these proteins wereabove 3 kDa larger than the authentic forms. Notwithstanding this theexpression products of the vectors containing the 5C variant of RNA 2(SEQ ID No: 51) are still useful because the resulting product, a P70(SEQ ID No: 52) variant, is only modified at the NH₂ terminus. Sincethis terminus is thought to be embedded in the capsid structure andtherefore not to participate in the initial interaction with the larvalmidgut cell, the variant is still useful.

In order to produce constructs which ensure that the expressed proteinspossessed the native amino terminus, new plasmids carrying the correctsequence were then cloned into the expression vector (pT7T2). It wasfound these plasmids to express proteins of the correct size.

The P6 has not yet been to expressed from the new constructs. Noevidence has been found for processing of P70 to yield the matureproteins in bacteria, nor upon in vitro translation of syntheticfull-length RNA 2 (SEQ ID No: 47).

The P17 (SEQ ID No: 48) gene has also been cloned into the same vectorsfor expression and bio-assay. This protein accumulates well in bacteriaupon induction, and electron microscopy analysis has shown it formspectacular honeycomb-like structures under the bacterial cell wall,completely surrounding the cell interior (results not shown). Theproperties of this protein including its amino acid composition andability to form tube-like structures when expressed in bacteria suggestthat it may be an homolog of a gap junction protein. The latter isinvolved in forming the channels linking the cytoplasms of adjacentepithelial cells in the insect gut. P17 could then play a role inenlarging or forming these channels, thereby enabling cell-to-cellmovement of the virus in the insect gut, analogous to the movement orspreading proteins encoded by plant RNA viruses.

In order to ensure that the expressed proteins carried the native aminoterminus the correct sequence has also been cloned into the expressionvector (pT7T2). The vector had been very slightly modified to thatdescribed above to introduce two novel restriction sited (for AflII andBgIII) flanking the Shine-Dalgarno sequence. The resulting constructshave been found to be poor producers of the capsid proteins. Thecomplete coding regions (which have been completely checked byre-sequencing) have therefore been recloned into the more satisfactoryvectors. Results using these constructs suggest that the amino-terminusof the capsid protein presents inherent difficulties in expression.These difficulties may be imposed by either the nucleotide sequenceencoding the amino terminus, or the actual amino acid sequence itself.To discriminate between these possibilities, two types of mutants weremade in the sequence encoding the amino terminal 5 residues of the HaSVcapsid protein. These amino-terminal mutants are as follows: HVP71GLYCCCATATG GGC GAT GCC GGC GTC GCG TCA CAG (SEQ ID No: 28) Met Gly Asp AlaGly Val Ala Ser Gln (SEQ ID No: 29) HVP71SER: CCCATATG AGC GAG GCC GGCGTC GCG TCA CAG (SEQ ID No: 30) Met Ser Glu Ala Gly Val Ala Ser Gln (SEQID No: 31) Native HaSV seq: ATG GGA GAT GCT GGA GTG GCG TCA CAG (SEQ IDNo: 32) Met Gly Asp Ala Gly Val Ala Ser Gln (SEQ ID No: 33)

EXAMPLE 4 Expression in Baculovirus Vectors and Bioassay on Larvae

Materials and Methods

A(i) Cloning of HaSV Capsid Protein Gene.

The capsid protein gene was amplified by PCR using the followingprimers: 5′ primers: (SEQ ID No: 34) HV17V71:5′ GGGGGATCCCGCGGATTTATGAGCGAG (SEQ ID No: 35) HV17E71:5′ GGGGGATCCCGCGGAGACATGAGCGAGCACAC (SEQ ID No: 36) HVP71:5′ GGGGGATCCAGCGACATGAGAGATGCTGGAGTGG (SEQ ID No: 37) HVV71:5′ GGGGGATCCAGCGACATGAGAGATGCTGGAGTGG

The ATG triplets initiating P17 (SEQ ID No: 48) (in HVI7V71 (SEQ ID No:34) and HV17E71 (SEQ ID No: 35)) or P71 (SEQ ID No: 50) (in HVP71 andHVV71) are underlined)

3′ Primers:

Primers HVP65C (SEQ ID No: 19) and HVP6C2 (SEQ ID No: 20), described inExample 3. Results section Xiiia, were used. These constructs were madeusing one of the four 5′ primers and HVP6C2 (SEQ ID No: 20). Plasmidsconstructed from PCR products made using one of the four 5′- primers andHVP65C (SEQ ID No: 19) are called 17V64 (made using 5′ primer 17E71 (SEQID No: 35)), P64 (made using 5′ primer P71 (SEQ ID No: 36)) and V64(made using 5′ primer V71 (SEQ ID No: 37)). These plasmids allowexpression of P64.

A(ii) Cloning a Full Length eDNA of HaSV RNA 1 (SEQ ID No: 39).

For expression of an RNA transcript corresponding to full length HaSVRNA 1 (SEQ ID No: 39), in insect cells by baculovirus infection orplasmid transfection, PCR was used to generate a fragment of cDNAlinking the 5′ end of RNA 1 (SEQ ID No: 39) to a Bam HI site.

The primers were: (SEQ ID No: 38) HVR1B5′ 5′ GGGGGATCCGTTCTGCCTCCCCGGAC(where the underlined nucleotide represents the start of natural RNA 1(SEQ ID No: 39)), and an oligonucleotide complementary to nucleotides1192=1212 of RNA 1 (SEQ ID No: 39).The template was plasmid pSRl(E3)B described in Example 3 above.A segment of the 1240 bp PCR fragment corresponding to the 5′ 320nucleotides of RNA 1 (SEQ ID No: 39) was excised with Bam HI and ASC IIand cloned into the Bam HI site of pBSSK(−)[Stratagene] together withthe 5 kbp ASCII—Bam HI fragment of pSR1(E3)B, giving plasmid pBHVR1B,which carries the complete cDNA to HaSV RNA 1 (SEQ ID No: 39), flankedby BamHI sites.A(iii) Cloning a Full Length CDNA of HaSV RNA 2 (SEQ ID No: 47).

For expression of an RNA transcript corresponding to full length RNA 2(SEQ ID No: 47) in insect cells by baculovirus infection or plasmidtransfection, plasmid pB+NR2B was made by inserting a fragment carryingHind III and Bam HI sites from the multiple cloning site of vectorpBSSK(−) [Stratagene] into plasmid pSR2B described above. The resultingplasmid, called pBHVR2B, carried the cDNA corresponding to full lengthHaSV RNA 2 (SEQ ID No: 47), flanked by Bam HI sites.

A(iv) Baculovirus Transfer Plasmids.

Bam HI fragments of 5.3 and 2.5 kbp corresponding to HaSV RNA's 1 and 2(SEQ ID Nos: 39 and 47) respectively, were excised from pBHVR1B andpBHVR2B respectively and inserted into the baculovirus transfer vectorsdescribed below, which had been linearised with Bam HI.

B. Baculovirus Expression of Proteins.

Baculovirus transfer vectors and engineered AcMNPV virus weretransfected into Spodoptera frugiperda (SF9) cells as described by thesupplier (Clontech) and as described in the following references:

Vlak, J. M. & Kens, R. J. A. (1990) in ‘Viral Vaccines”, Wiley-LissInc., NY, pp. 92-128; Kitts, P. A. et al (1990) Nucleic Acids Research18: 5667-5672; Kitts, P. A. and Possee, R. P. (in preparation); Possee,R. D. (1986) Virus Research, 5: 43-59.

Western Blotting.

As in Example 1

Oligonucleotides.

The following Ribozyme Oligonucleotides were produced according tostandard methods. HVR1Cla (SEQ ID No: 5)5′ CCATCGATGCCGGACTGGTATCCCAGGGGG 5′ HVR2Cla (SEQ ID No: 6)5′ CCATCGATGCCGGACTGGTATCCCGAGGGAC RZHDV1 (SEQ ID No: 7)5′ CCATCGATGATCCAGCCTCCTCGCGGCGCCGGATGGGCA RZHDV2 (SEQ ID No: 8)5′ GCTCTAGATCCATTCGCCATCCGAAGATGCCCATCCGGC RZHC1 (SEQ ID No: 9)5′ CCATCGATTTATGCCGAGAAGGTAACCAGAGAAACACAC RZHC2 (SEQ ID No: 10)5′ GCTCTAGACCAGGTAATATACCACAACGTGTGTTTCTCTResults

A series of recombinant baculoviruses has been constructed, based on thepVL941 transfer vector (PharMingen) or pBakPak8 (Clontech) and theAcMNPV. These are designed to express the correct forms of the precursorand processed HaSV capsid proteins (P64 and P71 (SEQ ID No: 50)) as wellas the smaller capsid protein P6, and P17 (SEQ ID No: 48). In allsystems where replicatable RNA encoding the nucleotide sequences of thepresent invention are to be used, such as eukaryotic systems, in orderto get efficient replication, translation or encapsidation of the RNA itis necessary to excise structures downstream of the t-RNA like structuresuch as the 3′ extension or poly A tail on the RNA. In order to carryout such an excision, ribozymes or other suitable mechanisms may beemployed. This self cleavage activity of the ribozyme containingtranscript should proceed at such a rate that most of the transcript istransported into the cytoplasm of the cell before the regeneration of areplicatable 3′ end occurs. Such ribozyme systems are more fullyexplained in Examples 7 and 9. In the results presented here highlyefficient production of P64 and P71 (SEQ ID No: 50) has been achieved.Electron microscopy and density gradient analysis have confirmed thatempty particles (“capsoids”) are being produced in infected cells thatefficiently express the P71 precursor gene. P17 (SEQ ID No: 48) placedin the context of the H. virescens juvenile hormone esterase (JHE) gene(Hanzlik T. N., et al, J. Biol. Chem. 264, 12419-25 (1989)) is produced,but not in large amounts. The latter construct results in a reduction ofexpression of the capsid protein from the same recombinant, presumablydue to a reduction in the number of ribosomes reaching the AUG for thecapsid gene.

SF9 cells infected with recombinant baculovirus have been shown tocontain large amounts of icosahedral virus particles by electronmicroscopy (data not shown). These particles contained no RNA, and wereempty inside. This observation shows that signals on the viral RNArequired for encapsidation of RNA must be located in either the 5′ 270nucleotides or the 3′ 170 nucleotides, or both, since these sequenceswere missing from the RNA transcripts made using recombinantbaculovirus. Expression of HaSV proteins was confirmed by Westernblotting of total protein extracts from infected insect cells.

In addition, the pAcUW31 vector (Clontech), which carries two promoters,is being used to simultaneously express p6 and p64 as separate proteins.

In order to bioassay the capsid protein produced in baculovirus infectedcells, it is first necessary to purify it from the baculovirusexpression vector. Preliminary attempts have made use of densitygradients, based on the observation that empty virus particles(“assembled capsids”) are in fact produced in infected cells.

As outlined earlier, the HaSV genome or portion thereof is aparticularly effective insecticidal agent for insertion into baculovirusvectors. Such a vector is constructed by insertion of the complete virusgenome or portion thereof (preferably the replicase gene) into thebaculovirus genome as shown in FIG. 13. Preferably the virus genome orreplicase is transcribed from a promoter active constitutively in insectcells or active at early stages upon baculovirus infection. An exampleof such a promoter is the heat shock promoter described in Example 7.Heat shock promoters are also activated in stressed cells, for examplecells stressed by baculovirus infection. An even more preferable use ofsuch a baculovirus construct is to use the HSP promoter to drive theHaSV replicase and another gene for a toxin (as exemplified elsewhere inthe specification) where the RNA expressing the toxin gene is capable ofbeing replicated by the HaSV replicase. Such recombinant baculovirusescarrying the HaSV genome or portions thereof for expression in larvae atearly or other stages of the baculovirus infection cycle areparticularly effective biological insecticides.

EXAMPLE 5 Effect of HaSV Genes and their Products on Plants

Materials and Methods

A. Electroporation of Protoplasts.

Protoplasts of Nicotiana tobacum, N. plumbaginifolia and Triticumaesticum and oats were produced and electroporated with either HaSV orHaSV RNA as described in Matsunaga et al (1992) J.Gen. Virol. 73:763-766.

B. Northern Blot Analysis—RNA Extraction from Protoplasts After Harvest

The protoplasts are subjected to 3 cycles of freezing and thawing, andthen an equal volume of 2× extraction buffer (100 mM Tris-HCl, pH 7.5,25 mM EDTA, 1% SDS, made in DEPC treated water) is added, followed by 1volume of phenol (equilibrated in 10 mM Tris-HCl pH 8.0) heated to 65

C. The samples are mixed by vortexing and incubated at 65

C for 15 min, vortexing every 5 min. After phase separation bycentrifugation at room temperature for 5 min, the aqueous phase isre-extracted with phenol, re separated by centrifugation andre-extracted with chloroform/isoamyl alcohol. To the aqueous phase arethen added 0.1 volume of DEPC-treated sodium acetate (pH 5.0) and 2volumes of ethanol. The RNA is recovered by precipitation at −70° C.,followed by centrifugation at 4° C. for 15 min. The samples were thenanalysed by agarose gel electrophoresis as described in example 1.

After blotting to Zeta-Probe membrane (BioRad), the hybridizationprotocols were as above for Example 2.

C. Total Protein from HaSV—Electroporated Protoplasts.

Protoplasts were analysed by SDS-polyacrylamide gel electrophoresis andWestern blotting as described in Example 1.

Results

i) Use of Complete (Replication-Competent) RNA Virus Genome inProtoplasts

a) HaSV Replication in Protoplasts

The nodavirus FHV has previously been shown to replicate in barleyprotoplasts (Selling H. H., Allison, R. F. and Kaesberg, P. Proc. Natl.Acad. Sci. USA 87,434-8 (1990). To determine whether HaSV virus RNA canreplicate in plants protoplasts, when introduced by electroporation,experiments using protoplasts from Nicotiana plumbaginifoli and wheathave been conducted. (These are all species for which protoplasts areregularly available in the Division of Plant industry). Assays forreplication including RNA (Northern) blots using probes derived fromcloned fragments of cDNA to RNAs 1 and 2 (SEQ ID Nos: 39 and 47), andWestern blots, using the antiserum to purified HaSV particles. Initialexperiments showed that both HaSV virus and RNA electroporated intoprotoplasts of N. phimbaginifolia resulted in HaSV replication asstudied using and verified by northern blots and ELISA. As a positivecontrol TMV RNA was electroporated and was replication observed.

b) Bioassays

Protoplasts into which HaSV RNA had been introduced by electroporationwere harvested after 6 or 7 days post electroporation and used inbioassays on neonate larvae by addition to normal diet. The resultsshowed significant stunting of test larvae in comparison to controllarvae (see Table 1 below). Protoplasts lacking HaSV RNAs had no effecton the larvae, confirming the result of control experiments. This resultconfirms that HaSV RNA, when expressed or replicated. in plant cells, isable to cause the formation of infectious virus particles able tocontrol insect larvae feeding on the plant material.

Northern blotting has been used to confirm that RNA electroporation intoprotoplasts leads to RNA replication. TABLE 1 Results of Bioassay from atypical experiment with Nicotiana and oat protoplasts (oat results areshown in brackets) [see over] Number Treatment Number Escapes stunted 1.diet only 12 (12) 2 (3)  0/10 (0/9) 2. diet + protoplasts 12 (12) 0 (1) 0/12 (0/11) 3. HaSV + diet 12 (12) 0 (1) 12/12 (11/11) 4. diet +HaSV/protoplasts 12 (n.d.) 0 (n.d.) 12/12 (n.d.) 5. diet +RNA/protoplasts 12 (12) 0 (0) 11/12 (10*/12)HaSv replication in the larvae was confirmed except for two larvae whichwere dead.The letters “n.d.” mean the experiment was not done.

The above results demonstrate assembly of HaSV particles fromelectroporated RNA in protoplasts of both moncot and dicot plantspecies.

c.) Plasmids to Test Replication of Cloned and Engineered Forms of HaSV

(1) Plasmids allowing in vitro transcription of HaSV RNAs 1 and 2 (SEQID Nos: 39 and 47) for electroporation into protoplasts have alreadybeen described above.

(2) Plasmids for transient expression of individual HaSV RNAs (1 or 2)(SEQ ID Nos: 39 and 47) in protoplasts. Full-length cDNAs for the twoviral RNAs have been inserted into expression plasmids pDH51 (with theCaMV 35 S promoter. Pietrzak M., et al (9186) Nucl. Acids Res. 14,5857-68) for dicots and pActl.cas (with the rice actin promoter) formonocots (McElroy et al (1990) The Plant Cell 2: 163-171). As with thevectors for expression in insect cells, these expression plasmids arebeing modified to include a cis-acting ribozyme for generation ofauthentic ends. The non-ribozyme plasmids gave no virus replication.

ii) Expression of Capsid Protein in Plants

In view of the present inventors' observation that empty particles(“assembled capsids”) are being produced in baculovirus-infected cellsthat efficiently express the P71 precursor gene, expression of thecoding region for the capsid protein in tobacco plants was investigated.The vector chosen for this purpose is based on pDH51 which carries theCaMV 35S promoter and polyadenylation signal. If necessary for improvedexpression, this vector can be modified by the addition of a translationenhancer sequence from e.g. TMV. Although certain groups haveconstructed transgenic plants expressing the capsid proteins of plantviruses, there has been only one recent report of assembly of emptycapsids in such plants (Bertioli et al.,(l991) J. gen. Virol. 72:1801-9). Bertioli et al point out that the protein-protein interactionsin most icosohedral plant RNA viruses may be too weak to allow assemblyof such capsids. In addition to the present inventors' observation ofempty HaSV capsids, it has been found these capsids are very tough,showing great resilience to e.g. repeated cycles of freezing andthawing, so that it is expected to see assembly of empty HaSV capsids(“assembled capsids”) in transgenic plants.

Construction of Capsid Protein Expression Plasmid.

Vector used was pDH51; linearised with BamHI and phosphatased. Insertwas PCR product made using following 2 primers: CAPPLANT:5′ GGGGATCC ACA ATG GGA GAT GCT GGA GTC-3′(BamHI)(i.e. A BamHI site followed by plant consensus context for ATG of capsidprotein gene and 15 further nucleotides of this gene—nts 366-383 of HaSVRNA2).HVP6C2 (Example 3)

The PCR product was made with VENT polymer (New England Biolabs). Aftergel purification, it was cut with BamHI and cloned into the vector.Orientation screened with EcoRI to identify insert in same direction aspromoter giving plasmid pDHVCAPB. Expression was verified by Westernblotting using anti-HaSV antiserum. Both precursor P71 and processed P64capsid protein were detected in protoplasts following transfection withpDHVCAPB, showing assembly of virus-like particles.

EXAMPLE 6 Identification of Midgut Binding Domains

Materials & Methods

A. Plasmid Construction

Was as described in Examples 3 and 4.

B. Western Blotting

Was as described in Examples 1 and 3.

C. Invitro Translation

In vitro transcripts of cloned CDNA of HaSV RNA's was translated invitro as in Examples 1 and 3.

D. Preparation of Brush Border Membrane Vesicles.

Brush Border Membrane Vesicles were prepared from freshly isolatedlarvae midguts of H.Armigera by the method of M.Wolfersberger et al(1987) Comp. Biochem. Physiol. 86A: 301-308, as modified byS.F.Garczyuski et.al. (1991) Applied Environ. Micro-biol 57: 1816-2820.Brush Border Membrane Vesicles binding assays using invitro labelledprotein or 125 I-labelled protein were as described in Garczynski et.al.(1991) or in H. M. Horton and Burand, J. P. (1993) J.Virol. 67:1860-1868.

Results

i) Determination of Epitopes on the Capsid Surface

Comparison of the recently published sequence of the Nudaurelia w virus(NwV) capsid protein with that of HaSV shown that these proteins areclosely related and fall into four distinct domains, which arealternatively variable and highly conserved. These domains aresummarised as follows: Residues: HaSV 1-49 50-272 273-435 437-647 NwV1-46 47-269 270-430 431-645 % identity: 37 81 34 81

Comparison of this observation with the alignment by Agrawal and Johnson(1992) between the NwV and the nodavirus BBV (whose crystal structure isknown: Hosur et al (1987) Proteins: Structure, Function & Genetics 2:167-176) showed that the variable region coincided with a region formingthe most prominent surface protrusion on the BBV capsid. Both HaSV andNwV carry large insertions at this point relative to BBV, and theseinsertions are largely different in sequence. Assuming that thealignment by Agrawal and Johnson (1992) is correct, then this means thatHaSV and NwV have a more prominent pyramid-like structures as a surfaceprotrusion than do the nodaviruses, and the pyramid-like structures aredifferent. As already noted, there is no immunological cross-reactivitybetween the two viruses, despite the high degree of identity. There isthus a strong implication of the variable domain as a surface protrusionwhich functions as the sole antigenic region.

To confirm this a 400 bp NarI fragment spanning the variable region wasdeleted from the capsid gene in the expression vector. With end-fillingof these sites the deletion is in-frame, so that a truncated protein ofca. 57 KDa is produced in bacteria upon induction. This protein wasrecognized only poorly on Western blots by the antiserum against intactHaSV particles made in rabbits. The central variable domain wasrecognized well by the antiserum when expressed in isolation from therest of the capsid gene.

As shown in the table above the region of HaSV capsid protein comprisingresidues 273-439 shows great divergence form the corresponding region ofthe NwV capsid protein, compared to its immediate flanking regions.Within this region an especially divergent domain is found from residue351 to residue 411, which shows only 25% identity to the correspondingregion of the NwV capsid protein. This region is flanked by thesequences corresponding to the b-sheet structural features b-E(residues339-349) and b-F(residues 424-431) of the HaSV capsid protein, based onthe alignment the NwV and nodavirus capsid proteins by Agrawal andJohnson (1992), and is therefore likely to form the loop of the mostprominent surface protrusion on the HaSV capsid. This is based oncomparison and correspondence to the nodavirus capsid protein structureand capsid structure as described by Wery J. -P. and Johnson, J. E.(1989) Analytical Chemistry 61, 1341A-1350A and Kaesberg, P., et al.(1990) J. Mol. Biol. 214, 423-435. This loop is thought to containimportant epitopes. It is significant that this exterior loop on thenodavirus capsid protein is one of the most variable regions when capsidproteins sequences from a number of nodaviruses are compared (Kaesberget al. 1990).

Finally, the present inventors have observed a significant level ofimmunological cross-reaction on Western blots, between antisera againstthe CryIA(c) Bt toxin and HaSV capsid protein, whether obtained fromvirus or expressed in bacteria. Initial data from the Narn deletionmutant described above suggest that this binding is not to the centralvariable domain, but to other regions of the capsid protein. The onlyother region of the proteins which shows extensive sequence variability,the amino terminus, cannot be responsible for the binding, since bothauthentic capsid protein and the protein with an altered amino terminusexpressed in bacteria are recognized by the anti Bt antisera.

ii) In-Vitro Binding Assays

The full-length clones for in vitro translation yielding highly ³⁵S or³H labelled proteins were constructed by replacing the bacterialtranslation interaction signal in the T7 plasmids above by the moreactive eucaryotic context sequence from the JHE gene. The labelledcapsid protein made by in vitro translation of the in vitro transcriptsmay be tested for binding to brush border membrane vesicles (BBMV's).Conditions are optimised by testing different procedures. The deletionmutant lacking approximately 125 amino acids in the central region, andcontaining the variable domain, as well as others derived from it arealso tested.

iii) Fusion Proteins Comprising Virus Capsid Midgut Binding Domains andOther Proteins

The idea behind these tests is to fuse the binding domain from the HaSVcapsid protein to either large proteins (preferably indigestible,causing protein to aggregate in or on the midgut cells) or toxin domainsfrom other proteins with suitable properties but normally differentbinding specificities (e.g. Bt). In initial experiments, the gene forthe complete capsid protein has been fuised to the GUS gene, as has adeletion mutant containing essentially only the central portion of thecapsid gene. The resulting fusion proteins are being expressed inbacteria and tested for GUS activity, and makes them sensitive probesfor binding experiments on midgut tissue.

iv) Mapping Binding Sites Using Bt/HaSV Fusion Proteins

Analysis of deletion mutants of the CryIA(c) Bt toxin has identifieddomains which may be involved in determining the host-specificity ofthis Bt by acting as receptor-binding sites (Schnepfet al (1990) J.Biol. Chem. 265: 20923-20930; Li et al (1991), Nature 353: 815-21. Thepresent inventors have obtained a clone of this toxin gene. Deletionmutants corresponding to those identified by Schnepf et al areconstructed. Segments of the HaSV capsid protein gene can then beinserted into these mutants, the protein expressed in bacteria and theirinsecticidal function assayed.

EXAMPLE 7 Viral Growth in Cell Culture

Materials & Methods

A. Cell Lines

The following cultured insect cell lines were tested for infection byHaSV: Drosophila melanogaster, Helicoverpa armigera (ovarian derived),Heliothis zea (ovarian derived), Plutella xylostella, Spodopterafrigiperda (SF9). All lines were grown under standard conditions. Uponreaching confluence, the culture medium was removed and all mono-layerscovered with 1.5 ml of cell culture medium into which HaSV had beendiluted; the average multiplicity of infection (M.O.I.) was 104. Afteradsorption at 26

C for 2h, the inoculum was removed, the cells carefully washed twicewith phosphate buffered saline (pH 7.0) and incubation continued with 5ml of 10%. Foetal calf serum in TC 199 culture medium (Cyto Systems).

B. Northern Blotting Analysis.

Virus replication in all the above cell lines was confirmed by northernblotting analysis. Total RNA was extracted from infected cells by themethod of Chomczynski and Sacchi (1987). Anal. Biochem. 162: 156-159.The cells were lysed in 1 ml of lysis solution (4M guanidiniumthiocyanate, 25mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1M2-mercaptoethanol). In order, 0.1 ml of 2M sodium acetate, pH 4, 1 ml ofphenol (0.2M sodium acetate equilibrated), and 0.2 ml ofchloroform-isoamyl alcohol mixture (49:1) were added with thoroughmixing between reagents. This was then vortexed for 10 s and cooled onice for 15 min. Tubes were centrifuged in an Eppendorf centrifuge at 14k for 15 min at 4

C for at least 15 min to allow RNA precipitation. RNA was pelleted bycentrifugation at 14 k for 15 min, washed with 0.6 ml of ice-cold 70%ethanol, pelleted once again (10K, 10 min), air dried at roomtemperature and resuspended in DEPC (Sigma) treated millipore water. RNAwas subject to denaturing agarose gel electrophoresis in the presence offormaldehyde according to Sambrook et.al. (1989). The gel was Northerntransferred to a zeta-probe membrane (Biorad) as described by Sambrooket.al. (1989). The probe was prepared by random-priming the 3′ sequencesof the HaSV genome using DNA and cDNA clones pSHVR15GB and pT7T2p7l SR-Ias per manufacturer's instructions (Boehringer-Mannheim). Hybridizationwas carried out as described for the standard DNA probe protocolcontained within the literature for the zeta-probe membrane (Biorad).

C. Vectors

Vectors as described below.

Results

It has been found that HaSV will replicate in several continuous celllines, of which the best is the Spodoptera frugiperda line SF9. Timecourse assays by Northern blotting in SF9 cells have shown that RNA 1(SEQ ID No: 39) replication is clearly detectable within a few hours ofinfection. RNA 2 (SEQ ID No: 47) is present only in very small amountsearly in infection and accumulates much more slowly than RNA 1 (SEQ IDNo: 39) does. This observation is consistent with one made earlier inHaSV-infected larvae, where RNA 2 (SEQ ID No: 47) replication was notobserved until 3 days after infection.

Some apparent replication was also observed in Drosophila cells (DL2),but with the difference that more RNA 2 (SEQ ID No: 47) replication wasobserved at the early time points compared to the lepidopteran celllines above.

Plasmids that express the HaSV genome as RNA transcripts from fulllength cDNA clones have been constructed and tested. These clones,constructed by PCR and carefully checked, have restriction sitedimmediately adjacent to the ends of the sequence. Transcription isdriven from a specially-re-engineered Drosophila HSP70 promoter.

i) Constructs for Expression in Insect Cells

The constructs are based on vectors carrying the Drosophila HSP 70 oractin promoters and suitable polyadenylation signals from Drosphila(Corces & Pellicer (1984) J. Biol. Chem. 259: 14812-14817) or SV40(Angelichio et al (1991) Nucl. Acids. Res. 18: 5037-5043). Sincetranscription from such plasmids generates viral RNAs carrying long 3′terminal extensions derived from sequences in the poladenylation signalfragment, it is necessary to achieve cleavage of the transcriptimmediately after the 3'sequence of the viral RNA. These plasmids gaveno virus replication, presumably because of the 3′ terminal extension.The method of choice for obtaining authentic 3′ termini is based onintroduction of DNA sequences encoding a cis-acting ribozyme into theconstructs. With suitable engineering, such a ribozyme will cleaveimmediately 3′ to the viral sequences within the transcript. Suitableribozymes, based on the hepatitis delta virus (Been M. D., Perrotta, A.T. & Rosenstein, S. P. Biochemistry 31, 11843-52 (1992) or the hairpincassette ribozyme (Altschuler, M., Tritz R. & Hampel, A. Gene 122, 85-90(1992) have been designed (see Example 4). This involves synthesis ofoverlapping oligonucleotides, which are then annealed and end-filledwith the Klenow fragment of DNA polymerase, to create short DNAfragments encoding the desired ribozyme. These fragments carryrestriction sites at their termini allowing them to be ligated intoplasmids between the viral RNA cDNA (which has a 3′ restriction siteadded by PCR) and the restriction fragment carrying the poladenylationsignal. Ribozyme function has been verified (Example 9).

The Drosophila HSP70 promoter was joined to the HaSV RNA 1 sequence asfollows. A BamHI restriction site was introduced into the promotersequence as described on p.5 of this specification. OligonucleotideHVR1B5P described in Example 8 was used to prime PCR of RNA 1 to yield acDNA copy of the RNA carrying a BamHI restriction site 5′ to the RNA 1sequence and separated from it by the nucleotides ACA which end theHSP70 promoter just before the start of transcription. This common BamHIsite was used to link the HSP70 promoter and the HaSV RNA 1 sequence.The resulting plasmid was completed by adding either the hairpincassette ribozyme (giving plasmid pHSPR1HC) or the HDV ribozyme (givingplasmid pHSPR1HDV) plus the SV40 late polyadenylation sequence.

A similar approach was used to obtain plasmids for RNA 2 i.e. pHSPR2HCand pHSPR2HDV.

An alternative approach is to link the promoter and the HaSV cDNAs usingblunt end ligation of a DNA fragment and carrying the promoter andterminating at the last nucleotide before the start of transcription(the underlined residue in ACA) and the cDNA fragments corresponding toeither HASV RNA 1 or 2, as described for the plant expression plasmidsin Example 9.

The latter approach was used to join the sarcoma virus (RSV) longterminal repeat (LTR) promoter to the HaSV cDNAs for expression ininsect cells. The RSV LTR promoter is active in many animal cells(Cullen, B. R. Raymond, K. & Ju, G. (1985) Mol. Cell. Biol. 5,438-447)and also in lepidopteran cell lines (D. Miller personal communication).It was obtained from plasmid pRSVCAT (Gorman, C., Padmanabhan, R. &Howard, B. H., (1983) Science 221, 551-553) as a 495 bp fragmentcarrying a 5′-XbaI site (added by PCR) and terminating at a blunt endwith the sequence AAC, with the underlined residue corresponding to thatimmediately before the start of transcription. The resulting plasmids,pRSVR1HCLA and pRSVR2HCLA, carry the HaSV RNA 1 and 2 cDNAs,respectively, and are otherwise like pHSPR1HC and pHSPR2HC,respectively. These plasmids carry the SV40 late polyadenylation signal.They allow efficient and precise expression of the HaSV genomic RNAs ininsect cells, for example if introduced using a baculovirus vector or bytransfection.

EXAMPLE 8 Shedding of Infected Cells

Materials & Methods

A. Confocal Laser Scanning Microscopy. (CLSM)

CLSM enables the visualisation and analysis of three-dimensional celland tissue structures at the macro and molecular levels. The Leica CLSMused in this example is based on an MC 68020/68881 VME bus (20MHz) withstandard 2Mbyte framestore and 4Mbyte RAM and OS9 operating system withprogrammes written in C code. It incorporates a Leica Diaplan researchmicroscope and using X10/0.45, X25/0.75,X40/1.30 and X63/1.30 Fluotarobjectives has a claimed optical efficiency better than 90%. Theconfocal pinhole is software controlled over the range of 20 to 200 mm.Excitation at 488 and 514 nm is provided by a 2 to 50 mW argon-ionlaser.

B. Immunocytochemistry (ICC).

For whole mount ICC, tissues were dissected under saline and fixed infresh 4% formaldehyde in phosphate buffered saline (PBS) for at least 15mins. After multiple washes in PBS they were permeablized either by 60mins incubation in PBT (PLBS with 0.1% Triton X-100 plus 0.2% bovineserum albumin). After 30 mins blocking in PBT+N (5% normal goat serum)tissue was incubated in primary antibody diluted (1:40) in PBT+N for atleast 2 hrs at room temperature then at 4

C overnight. After extensive washing in PBT and 30 mins blocking inPBT+N the FITC conjugated secondary antibody diluted (1:60) in PBT+N wasincubated for 2 hrs at room temperature plus overnight at 4

C. After multiple washes in PBT and PBS the tissue was cleared in 70%glycerol and mounted in 0.01% w/v p-phenylenediamine (Sigma#P1519)dissolved in 70% glycerol. All processing was at room temperature unlessotherwise stated.

Results

The inventors′ current model for the effect of HaSV involves thedetection by the insect midgut of infected cells, their identificationas infected and their subsequent shedding in numbers sufficient to causeirreparable damage to the insect midgut. The evidence for this is basedon the above and on the following direct observation of the fate ofinfected cells in midgut tissue over 1-3 days post infection. Theseresults in repeat experiments were complicated by the discovery thatanother unrelated virus was present in the larval population beingtested. Preliminary findings indicated that HaSV infection activates orfacilitates pathogenesis of the unrelated virus and together these causesevere disruption of the larval gut cells. Thus these two agents appearto act synergistically in causing gut cell disruption.

Midguts from larvae infected with HaSV were treated with the antiserumto purified HaSV particles (above) and examined under the Laser confocalmicroscope (described above). This established that some midgut cellswere sufficiently infected with HaSV to give strong fluorescencesignals. Such cells were moreover clearly separating from thesurrounding tissue, a sign that they were in the process of being shed.

Similar observation have been made with other insect viruses (Flipsen etal (1992) Society for Invertebrate Pathology Abstract #96) although inthese cases the effect is too localised and weak to cause anyanti-feeding effect apparently only the small RNA virus of thetetraviridae which are localised to the gut and cause more-or-lesssevere anti-feeding effects in their hosts (Moore, N. F. in Kurstak E.(Ed) (1991) Viruses of Invertebrates. Marcel Dekker, New York pp277-285) are capable of such an effect to an extent sufficient for pestcontrol.

Following on from the immune-fluorescence work, in situ hybridizationcan be carried out to detect RNA replication in infectedcells.Furthermore, larvae infected with a recombinant HaSV expressing aforeign gene at early stages (by insertion of that gene into RNA 1 inplace of the N-terminal portion of the replicase gene) can be studied. Acorrelation between virus replication and cell rejection can beconfirmed by histochemical analysis of the midgut cells of the infectedlarvae. Thus the cell-shedding phenomenon offers a direct and rapidassay for early events in HaSV-infected gut tissue. Extracts ofbaculo-vector infected insect cells carrying empty HaSV particles can befed to larvae directly and the midgut examined by toluidine bluestaining and immune-fluorescence at intervals after infection. This willallow direct determination of whether the particles can bind the brushborder membranes in intact gut, and whether such binding can induce themassive disruption evident in normally infected larvae. Controlexperiments using extracts from cells infected with the baculovectoralone can be conducted to observe and distinguish effects due to thevector. The immune-fluorescence assay on midgut tissue allows analysisof binding to midgut brushborder membranes. Once determined forwild-type capsid protein expressed from a baculo-vector, deletion orreplacement mutants can be inserted into the baculovectors. Suitablecell extracts from these can be used to infect larvae.

EXAMPLE 9 Engineered Virus and Uses

Materials & Methods

(as indicated in earlier Examples)

i) Engineered Virus as a Vector for Other Toxin Genes

This involves placing suitable genes under control of HaSV replicationand encapsidation signals. Genes which may be suitable includeintracellular insect toxins such as ricin, neurotoxins, gelonin anddiphtheria toxins. The toxin gene may be placed in the viral gene suchthat it is a silent (downstream) cistron on a polycistronic RNA, or in aminus strand orientation, requiring replication by the viral polymeraseto be expressed. Standard techniques in molecular biology can be used toengineer these vectors.

A discussion of two recombinant HaSV vectors which have been designed isgiven below:

for RNA 1 (SEQ ID No: 39):

The reporter gene (or one of the toxin genes mentioned above) isinserted in place of the amino-terminal portion of the putativereplicase gene, such that the intiation codon used for the replicase (iethat at nucleotides 37-39 of the sequence) is now used to commencereporter gene translation. The fusion is achieved by the use ofartificial NcoI restriction sites common to both sequences.

The short 36 nucleotide 5′-untranslated leader of RNA 1 (SEQ ID No: 39)(shown in upper case) is synthesised as the following sequence:ggggatccacaGTTCTGCCTCCCCCGGACGGTAAATATAGGGGAACCATG Gtctagagg, (SEQ IDNo: 53)

using two overlapping oligonucleotides comprising the first 31(oligonucleotide HVR1B5P) nucleotides and the complement of the last 40nucleotides (oligonucleotide HVR1NCO) respectively. These primers areannealed and end-filled by Klenow. The resulting fragment is then cutwith BamHI and XbaI (sites underlined) and cloned with plasmid vectorpBSIISK(−) to give pBSSKR1NCO.

The GUS gene carrying a Ncol site at the ATG codon was obtained as aNcoI-SacI fragment from plasmid pRAJ275 (Jefferson, RAJ Plant Mol. Biol.Rep 5, 3387-405 (1987)). This Sacd site is located just downstream fromthe coding sequence for the GUS gene.

The 5′ leader of HaSV RNA1 is excised as a BamHI-NcoI fragment from theplasmid pBSSKR1NCO, and is ligated together with the NcoI-SacI fragmentcarrying the GUS gene into plasmid pHSPR1HC or pHSPR1HDV or pDHStuR1HCcarrying the full-length cDNA insert of RNA 1 (see above) which has beencut with BamHI and SacI. The resulting plasmid then carries a completeform of RNA 1 (SEQ ID No: 39) but with the amino-terminal portion of thereplicase gene substituted by the GUS gene. It is desirable to produce aconstruct with approximately the same size as RNA 1 (SEQ ID No: 39) forencapsidation purposes.

Similar approaches are adopted for RNA 2 (SEQ ID No: 47), with theforeign, reporter or toxin gene fused to the initiation codon of eitherP17 or P71. In either case the context sequence of the introduced geneis modified to give the necessary expression level of that protein. Theforeign gene is introduced into plasmids pHSPR2HC or pHSPR2HDV orpDHStuR2HC.

The above recombinants have been described specifically as insertions ofa reporter gene (GUS). The toxin genes to be inserted are described onpage 14 of the specification. These preferably further require a signalpeptide sequence added at the amino-terminus of the protein.

ii) Capsid Technology

Identification of encapsidation (and replication) signals on virus RNAallows design of RNAs which can be encapsidated in HaSV particles duringassembly of virus in a suitable production system. The virus capsidsthen carry the RNA of choice into the insects midgut cells where the RNAcan perform its intended function. Examples of RNAs which may beencapsidated in this manner include RNAs for specific toxins such asintracellular toxins, such as ricin, gelonin, diptheria toxins orneurotoxins. This strategy is based on the resistance of the virusparticle to the harsh gut environment.

iii) Other Uses of the Capsid Particle

The capsid particles can be used as vectors for protein toxins.Knowledge of icosahedral particle structure elucidated by the inventorssuggests that the amino and especially the C-termini are present withinthe capsid interior. It is possible to replace or modify the amino acidsequence corresponding to P7 such that it encodes a suitable proteintoxin which is cleaved off the bulk of the capsid protein during capsidmaturation. As with toxin-encoding mRNAs, the HaSV capsid delivers it tothe midgut cell of the feeding insect, where it exerts the desired toxiceffect.

iv) Use of HaSV in Plants

The use of HaSV in the production of insect-resistant transgenic plantsare shown in FIG. 12. These inventions are based on the use of eitherthe complete HaSV genome, or of the replicase gene as a tool for theamplification of suitable amplifiable mRNAs (e.g. encoding toxin) or ofthe capsid protein as a means to deliver insecticidal agents. Thesestrategies are now described in some detail.

a) Use of the Complete HaSV Genome

Fragments of cDNA corresponding to the full-length HaSV genomecomponents RNAs 1 and 2 (SEQ ID Nos: 39 and 47) are placed in a suitablevector for plant transformation under the control of either aconstitutive plant promoter (e.g. the CaMV 35S promoter mentioned above)or an inducible promoter or a tissue specific (e.g. leaf-specific)promoter. The cDNAs are followed by a cis-cleaving ribozyme and asuitable plant polyadenylation signal. Transcription and translation ofthese genes in transgenic plant tissues and cells leads to assembly offully infectious virus particles to infect and kill feeding larvae.

The following experiments were conducted. The plasmids for expressionused the CaMV 35S promoter to generate transcripts commencing at thefirst nucleotide of the HaSV RNAs 1 and 2 (SEQ ID Nos: 39 and 47). Thevector pDH51 (M. Pietrzak, R. Shilito, T. Hohn and I. Potrykus (1986).Nucleic Acids Research 14, 5857) which carries the CaMV 35S promoterfollowed by a multiple cloning site and the CaMV polyadenylationfragment was modified to make a suitable vector, pDH51Stu, carrying aStuI site at the immediate 3′ end of the CaMV 35S promoter. The promoterthereby terminates in the sequence GAGAGGCCT, with the underlinedresidue being that at which transcription would start. (Similar vectorshave been described by Mori et al., J. General Virology 72, 243-246(1991) and Dessens and Lomonossoff, ibid 74, 889-892 (1993).) The StuIsite (AGG/CCT) is followed by a BamHI site (GGATCC). Cleavage of thisvector with StuI and BamHI generates a vector DNA molecule with oneblunt end (from StuI cleavage) and one sticky BamHI end. This allowsligation of cDNA molecules corresponding to the full-length HaSV genomicRNAs, and carrying a blunt end at the 5′ end of the full-length cDNA anda BamHI site after the 3′-end of the full-length cDNA.

Suitable cDNA fragments carrying a blunt end corresponding to the5′-terminal nucleotide of either RNA 1 or 2 (SEQ ID Nos: 39 and 47) weregenerated using PCR and an oligonucleotide primer corresponding to the5′-terminal first 18 nucleotides of the sequence of either RNA 1 (SEQ IDNo: 39) or RNA 2 (SEQ ID No: 47). The cDNA sequence corresponding to the3′ terminal sequences of either RNA 1 (SEQ ID No. 39) or RNA 2 (SEQ IDNo 47) were followed on these DNA fragments by sequences correspondingto one of the ribozymes whose sequences are shown in FIG. 8 and whoseconstruction is described in Example 7. The 3′-terminal sequencecorresponding to an XbaI site (TCTAGA) shown in these ribozyme sequenceswas followed on the suitable DNA fragments by a BamHI site, which uponcleavage with this enzyme yielded a sticky end capable of being ligatedinto the BamHI end of the vector cleaved as described above. There weretherefore a total of four suitable DNA fragments for insertion into thevector:

RNA 1 (SEQ ID No: 39) followed by the hairpin cassette (HC) ribozyme

RNA 1 (SEQ ID No: 39) followed by the hepatitis delta virus (HDV)ribozyme

RNA 2 (SEQ ID No: 47) followed by the hairpin (HC) ribozyme

RNA 2 (SEQ ID No: 47) followed by the hepatitis delta virus (HDV)ribozyme.

These four fragments were individually ligated into the vector pDH51 Stucleaved with Stul and BamHI to generate four distinct plasmids asfollows:

pDHStuR1HC

pDHStuR1HDV

pDHStuR2HC

pDHStuR2HDV

Transcription from the 35S promoter in these plasmids results in RNAscommencing at the first nucleotide of either the RNA 1 sequence (SEQ IDNo: 39) or RNA 2 sequence (SEQ ID No: 47) and terminating in the CaMVpolyadenylation fragment. Self-cleavage at the locations shown in FIG. 8by the cis-acting ribozymes obtained within these transcripts generatesRNA molecules with the 3′-termini corresponding to the natural virustermini.

After amplification and purification on CsCl gradients, thirty mg ofeach of these four plasmids was transfected by electroporation intoaliquots of two million N. plumbaginifolia protoplasts (as described inExample 5) either individually or in the combinations listed below:

pDHStuR1HC+pDHStuR2HC

pDHStuR1HDV+pDHStuR2HDV

The production of infectious HaSV particles within transfectedprotoplasts was then demonstrated by bioassay on heliothis larvae. Afterincubation at 25

C for 3-5 days, the protoplasts were recovered by low speedcentrifugation and applied directly to standard heliothis diet assurface contamination for bioassay as described in Example 1. Stuntingwas only observed when plasmids expressing HaSV RNA 1 (SEQ ID No: 39)and RNA 2 (SEQ ID No: 47) were co-transfected, and then only in the caseof those carrying the hairpin ribozyme to generate the viral 3′ ends(see Table 2). In contrast, constructs carrying the HDV ribozyme at the3′ end were not infectious. The reasons for this have not beendetermined. As expected, expression of RNA 1 or 2 (SEQ ID Nos: 39 and47) alone in protoplasts did not lead to the assembly of infectiousparticles. Western blot analysis of protoplasts transfected with the RNA2 (SEQ ID No: 47) constructs did show production of limited amounts ofthe capsid protein.

Suitable control experiments confirmed that larval stunting was due toHaSV particles generated de novo in the protoplasts. As shown in theTable 2, neither the protoplasts alone nor protoplasts mixed withplasmid DNA were capable of initiating stunting. TABLE 2 No. of No.Treatment larvae Escapes stunted 1. diet alone 24 0 0 2. diet + HaSV 240 24 3. diet + protoplasts 24 0 0 4. diet + pDHStuR1HC 24 0 0 5. diet +pDHStuR1HDV 24 0 0 6. diet + pDHStuR2HC 24 0 0 7. diet + pDHStuR2HDV 240 0 8. diet + pDHStuR1HC + pDHStuR2HC* 24 0 22 9. diet + pDHStuR1HDV +24 0 0 pDHStuR2HDV* 10. pDHStuR1HC + pDHStuR2HC 24 0 0 (but mixed withprotoplasts)*these plasmids were co-transfected with pDHVCAPB (see Example 5)

HaSV infection of stunted larvae was confirmed by dot-blotting of RNAusing HaSV specific probes. After weighing, larva were sacrificed andtotal RNA extracted as follows. Each larva was homogenised in thepresence of 260 ml deionised water, 24 ml 2M sodium acetate pH 4.0 and200 ml phenol equilibrated with 2M sodium acetate pH 4.0. Aftercentrifugation at 14 000 rpm for 15 min at 4

C, the supernatant (about 200 ml) was removed and extracted once with anequal volume of chloroform. After centrifugation, the supernatant (about200 ml) was mixed with 20 ml of sodium acetate and 400 ml of absoluteethanol. The precipitate after centrifugation was vacuum dried andredissolved in 5-10 ml of sterile, DEPC-treated water. For dot-blotting,the RNA was mixed with 70 ml of DEPC-treated water and 30 ml of 10 mMEDTA, 30 mM NaOH. HaSV RNA was determined and quantified by dot blotting(as described in Example 2) using a probe random primed DNA from clonescorresponding to the terminal 1000 nucleotides of RNA 1 and 2. Alllarvae recorded as stunted in the bioassays were found to carry HaSV andgive signals comparable to those of the larvae fed purified HaSVparticles (Table 2). To confirm that the larvae were infected with HaSV,ten aliquots of protoplasts were electroporated with plasmidspDHStuR1HC+pDHStuR2HC and the protoplasts fed (after incubation) to 150heliothis larvae. The larvae were allowed to grow for one week, uponwhich significant stunting was observed in 50% of the larvae, and viruswas then purified from these stunted larvae as described in Example 1.Analysis on CsCl gradients showed the production of distinct bandscharacteristic of HaSV; analysis of the virus particles found in thesebands by SDS-PAGE and Western blotting with HaSV antiserum confirmedtheir identity as authentic HaSV.

These results have therefore demonstrated that DNA plasmids capable ofexpressing the HaSV genome in plant cells have been constructed. Onceintroduced into the cells, the plasmids are transcribed to yield HaSVgenomic RNA which can drive the assembly of particles able to infectheliothis larvae by the normal oral route. These constructs havetherefore been shown to be suitable for use in engineering transgenicplants expressing HaSV.

A variation on this strategy is to remove from the cDNA for RNA 2 (SEQID No: 47) the fragments encoding RNA encapsidation and/or replicationsignals. This results in either the assembly in the plant cells of HaSVparticles carrying only RNA 1 (SEQ ID No: 39), or of HaSV particlescarrying RNA 1 (SEQ ID No: 39) and a form of RNA 2 (SEQ ID No: 47) whichcannot be replicated in the infected insect cell.

A further variation on this strategy is to modify the plant transgeneencoding RNA 2 (SEQ ID No: 47) so that it is still replicatable andencapsidatable, but no longer express functional capsid protein. HaSVcapsids made in such plant cells will then be capable of making both thereplicase and P17 (SEQ ID No: 48) in infected insect cells, but not ofassembling progeny virus particles therein (such as shown in FIG.12(d)). These measures confer inherent biological safety in the form ofcontainment on the use of such transgenic plant material.

(b) Use of Portions of HaSV Genome to Deliver Toxins to Insect Cells

This approach makes use of any of the systems described in (a) above.Plant cells contain an additional transgene encoding a suitableinsect-specific, intracellular toxin (as described above). Such a toxingene is expressed by plant RNA polymerase in either a positive or anegative sense (the latter is preferred) and in such a form that the RNAcan be encapsidated by HaSV capsid protein and/or replicated by the HaSVreplicase in infected insect cells (see FIGS. 12 a and 12 b)

Transgenic plants would contain two different transgenes, making eitherunmodified capsid protein precursor or a modified form in which most ofthe carboxyterminal protein P7 is replaced by a suitable insect-specifictoxin or one which is inactive as part of a fusion protein. (Gelonin orother ribosome-inactivating proteins, insect gut toxins or neurotoxinsmay be suitable here.) Expression from these two transgenes would beregulated so that only the required amounts of the modified andunmodified forms are made in the plant cell, and assembled in suchproportions into the capsoids. One way to modulate the production ofcapsotixin fusion proteins is to make translation of the carboxyterminaltoxin reading frame dependent on a translational frameshift orread-through of a termination codon. With an appropriate low frequencyof frame-shifting (eg 0.1-2%), it could even be sufficient to use asingle transgene, if it were possible to synthesise the P7 portion andthe toxin portion as overlapping genes. Upon assembly (which we havedemonstrated in insect cells using the baculovirus vectors) andmaturation, the protein precursors are cleaved and release the mature P7and the toxin, which remain within the capsoids. These proteins are notreleased until capsoid disassembly occurs in insect gut cells. Theprocessed form of the toxin is then able to kill the pest.

(c) HaSV Particles Devoid of Nucleic Acid Carrying One or More SuitableProtein Toxins and/or their mRNA

A protein toxin (or toxins) is expressed as a fusion with the capsidprotein. The fusion protein then assembles into capsid carrying thetoxin(s). These capsids present in the plant tissue exert an antifeedingeffect on insects attaching the plant.

EXAMPLE 10 Expression of HaSV in other Delivery Vectors

Materials & Methods

(as indicated in earlier Examples)

Constructs similar to those for plant expression are introduced intoyeast or bacteria by standard techniques. Virus particles are assembledfor either filly infectious virus or any of the modified or biologicallycontained forms described in Example 9. Microbes produced in suitablefermentation or culture facilities and carrying such forms of the virusare then delivered to the crop by spraying. The microbial cell wallprovides extra protection for the virus particles produced within themicrobe.

Well established techniques exist for culture and transformation ofyeast (Ausubel, F. M. et al. (eds) Current Protocols in MolecularBiology. J. Wiley & Sons, NY, 1989). An example of a yeast expressionvector is pBM272, which contains the URA3 selectable marker (Johnston,M. & Davies, R. W. Mol. Cell. BIol. 4, 1440-8, (1984); Stone, D. &Craig, E. Mol. Cell. Biol. 10, 1622-32 (1990). Another example of anexpression vector is pRJ28, carrying the Trp1 and Leu2 selectablemarkers.

Yeast has recently been shown to support replication of RNA repliconsderived from a plant RNA virus, brome mosaic virus (Janda, M. &Ahlquist, P. Cell 72, 961-70 (1993). Since the BMV replicase isdistantly related to that of HaSV, and the two viruses are likely toreplicate by similar strategies within cells, yeast cells probablycontain all the cellular factors required for HaSV to generateinfectious virus.

For bacteria, suitable expression vectors have been described above.

EXAMPLE 11 The Transvirus Approach for Insect Pest Control: MakingTransgenic Plants Expressing HaSV

1. Vector Construction

A special binary vector was constructed for transforming plants with theHaSV genome. This vector is based on pART27 (A. Gleave (1992) PlantMol.Biol.20, 1203-1207), which was modified to (1) carry an alternativeorigin of replication for the host Agrobacteriuin tumefaciens and (2)incorporate restriction sites in the multiple cloning site forrestriction enzymes AscI and PacI which recognise rare (8 bp) sequences.

For engineering the multiple cloning site, pART27 was cut with SpeI andNotI. Ten picomoles of each of the two oligos whose sequence follows(TOP and BOTTOM) were annealed in 10 microlitres of water (heated to 80°C. for 2 min and allowed to cool slowly to room temperature). The stickyends on these annealed oligonucleotides allowed the insert to be clonedinto pART27 (giving pART27mod) as described in Example No. 3 and 9.

Sequence of oligonucleotide: (SEQ ID NO: 54) TOP:5′-GGCCGCTTAATTAAGGATCCGGCGCGCCA-3′ (SEQ ID NO: 55) BOTTOM:3′-CGAATTAATTCCTAGGCCGCGCGGTGATC-5′

The PacI recognition sequence is TTAATTAA ,SEQ ID NO: 56 and that forAscI is GGCGCGCC, SEQ ID NO: 57). A 4 kbp Sall fragment from plasmidpART27mod (containing the right border, lacZ marker (+multiple cloningsite)nptll gene for kanamycin resistance under control of the nospromoter and polyadenylation signal and the left border) was cloned intothe 13 kbp vector pKT231 linearised with XhoI. Plasmid pKT231 carriesthe IncQ origin of replication for the host Agrobacterium tumefaciensand a resistance (marker) gene for streptomycin/spectinomycin.(Bagdasarian, M. & Timmis, K. N. (1982) Curr. Topics Microbiol. Immunol.96, 46-67). These two fragments were ligated using standard protocols(eg in Example No 3) and transformed into E.coli strain DH5α usingstandard protocols (eg in Example No 3). The resultant plasmid was namedpJDML1.

2. Cloning HaSV Genes into Transfer Plasmid

Construction of Transfer Vectors with HaSV Genes.

Before the HaSV gene cassettes could be cloned into binary transfervectors pART27 mod or pJDML1, they were re-cloned into the vectorplasmid pBJ33 to provide flanking AscI and PacI sites. Plasmid pBJ33(provided by Bart Janssen) is based on pBC SK(+) supplied byStratagene), but with a multiple cloning site modified to contain thefollowing sites:

SacI/PacI/AscI/SacII/XbaI/SpeI/BamHI/PstI/EcoRI/EcoRV/HindIII/ClaI/SaII/XhoI/ApaI/PacI/AscI/KpnI.

The cDNA fragment corresponding to complete HaSV RNA 1 behind the 35Spromoter and terminating in the hairpin cassette ribozyme and the CaMVpolyadenylation signal fragment (approx 6 kpb in total) was excised fromplasmid pDHStuR1HC (Example 9) with EcoRI and cloned into EcoRI-cutvector pBJ33 to give plasmid pBJ33R1HC. Similarly, the cDNA fragmentcorresponding to complete HaSV RNA 2 behind the 35S promoter andterminating in the hairpin cassette ribozyme and the CaMVpolyadenylation signal fragment (approx 3.3 kbp in total) was excisedfrom plasmid pDHStuR2HC (Example 9) as two fragments, one (covering the35S promoter and the first 500 bp of the RNA 2 sequence) of about 1 kbpwith EcoRI and R5rII and the second (covering the remainder of the RNA 2sequence, the ribozyme and the polyadenylation signal) of about 2.3 kbpwith RerII and HindIlI. These two fragments were simultaneously ligatedinto EcoRI and HindIII-cut vector pBJ33 to give plasmid pBJ33R2HC.

A 1.9 kbp fragment comprising the 5′ 1.7 kbp of the HaSV capsid gene,together with the polyadenylation fragment, were excised from expressionplasmid pDHVCAPB (described in Example 5) as a EcoRI—KpnI fragment andcloned into pTZ19U (pharmacia) cut with EcoRI and KpnI, givingpTZ19UEVCAPB., This portion of the HaSV capsid gene expression cassettewas then re-excised as a HindIII-EcoRI fragment and cloned into PBJ33cut with these enzymes. This plasmid (pBJ33EVCAPB) was then linearizedwith EcoRI and the ca. 800 bp EcoRI fragment from pDHVCAPB carrying the35S promoter and the 5′ 250 bp of the capsid gene inserted, followed byscreening for orientation. The resulting plasmid carrying thereassembled complete capsid gene expression cassette was namedpBJ33VCAPB.

Assembling Binary Plasmids.

The RNA 1 expression cassette was excised from plasmid pBJ33R1HC withAscI and PacI and cloned into pART27 mod cut with AscI and PacI to givepMLR1. The RNA 2 expression cassette was also cloned as an AscI-PacIfragment into pJDML1 cut with AscI and PacI to give pJDMLR2.

The capsid protein gene cassette was excised from pBJ33 VCAPB with PacIand cloned into plasmid pMLR1 cut with PacI. Resulting plasmids werescreened for orientation and the plasmid with the capsid gene and RNA1in the same orientation was named pMLR1V. The complete fragment carryingthe HaSV capsid gene and RNA 1 expression cassettes in pMLR1V wasexcised with AscI and cloned into pJDMLR2 linearised with AscI to givepHaSV1 (29 kpb). This plasmid carries the HaSV capsid gene expressioncassette and the HaSV RNA 1 and RNA 2 expression cassettes in this orderand all in the same orientation. The kanamycin resistance gene islocated upstream of the capsid gene and in the opposite orientation.

Table of constructs generated: #Plants (independent Vector Insert(s)Name transformants) Comments pART27mod RNA 1 pMLR1 15 control pJDML1R1 + R2 + pHaSV1 30 complete CAP virus pART27mod R1 + CAP pMLR1V 15subvirus pJDML1 R1 + CAP pJDMLR1V 30 subvirus pART27mod RNA2 pMLR2 15control pJDML1 RNA2 pJDMLR2 15 control pART27mod CAP pMLVF 15 control(CAP = HaSV capsid gene)3. Plant Transformation and Regeneration

Binary transfer vectors (above) were transformed into Agrobacteriumtumefaciens strain LBA4404 by electroporation (Lin, J. J. (1994) FOCUS16,18-19; Lin, J. J. (1994) Plant Science 101, 11-15). Leaf discs fromNicotiana tabacum grown under sterile conditions were transformed usingcocultivation with transformed A. tumefaciens (Horsch, R. B. et al.(1984) Science 23, 496-498; Horsch, R. B. et al. (1988) Plant MolecularBiology Manual A5:1-9; as modified by Lisa Molvig (pers. comm.)) andgrown on kanamycin. After transfer of regenerating shoots for furtherselection on kanamycin medium, kanamycin-resistant roots were selectedand then tissue from these plants used to verify HaSV gene expression.The numbers of plants selected are shown in the table above for each ofthe constructs.

4. Western, Northern and Southern Blotting on Transgenic Plants

For western blots: A small amount (0.1 g) of fresh leaf material fromeach plant was extracted by grinding in 0.2 ml of plant extractionbuffer (0.2M NaCI, 0.1 M Tes, pH 7.65, 1 mM PMSF, 2% b-mercaptoethanol,lmM EDTA). After centrifugation to pellet plant debris the supernatantwas collected and 10_(—)1 aliquots run on a SDS-gel for blotting andimmuno-analysis with antibody against HaSV as described in Example 1.The results for the first plants assayed are given in Table 3.

For Northern blots: Total leaf RNA was extracted from 0.15 g of freshleaf material. The leaf material was ground under liquid nitrogen to apowder and then extracted by further grinding in 0.45 ml NTES buffer(0.1 M NaCl, 10 mM Tris-HCl pH 8.0, lmM EDTA, 0.1% SDS) plus 0.45 mlTris pH8.0-saturated phenol/chloroform. The slurry was vortexed,centrifuged for 3 min and the aqueous phase mixed with 1 volume ofisopropanol to precipitate RNA and DNA. After resuspending the pellet in0.1 ml water, 1 volume of 4M LiCl was added and the mix stood on iceovernight before centrifugation to pellet RNA. The RNA was then analysedby gel electrophoresis according to the methods in Examples 1 and 2.HaSV specific RNAs were detected by Northern blottings as described inExample 2 and by using riboprobes made to detect the 3′-terminal 1000nucleotides of each of RNA 1 and 2, made using the Promega Riboprobe kitand used as specified by the supplier.

For Southern blots: to detect HaSV genes in plant genomic DNA.

To recover plant genomic DNA, the supernatant from LiCI precipitation(above) was mixed with 2 volumes of ethanol. The pellet was redissolvedand the DNA cut with BamHI before analysis on agarose gels and transferto nylon membrane as described by Sambrook et al (1989) and by themanufacturer (Zetaprobe/BioRad).

HaSV-specific bands were detected described above.

5. Bioassays on Leaf Material

Two small leaves (2-3 cm in length) were selected from each transformedplant selected, and placed in petri dishes containing 1.5% agarose inwater. Three to 8 neonate larvae were placed in each petri dish andobserved for 3 days. At the end of this time, larvae were weighed andthen total RNA extracted as described in Example 1. The extent of leafdamage was quantified by measuring the area of leaf consumed by eachgroup of larvae over the three days of the assay (see Table 3). TABLE 3Preliminary bioassay of HaSV transgenic plants Three to 8 larvae wereplaced on a small leaf (from a newly regenerated plant) in a petri dishwith no provision of fresh food, after 3 days, larvae were sacrificedand northern blotted; also, protein extracted from leaves of the plantswere western blotted using anti-HaSV antisera. Western Blot Leaf forHaSV Northern Damage capsid blot for HaSV Larval (mm² Transformationprotein in RNA in Weight consumed/ Plant Plasmid plant (+/−) plant (mg)larvae) Negative Controls — — — 61 1.1 (subvirus) pJDMLR1V 0 1.1 ± 0.229 (RNA1 = p71) 3.2 (whole virus) pHaSV1 0 1.0 ± 0.4 38 3.4 (wholevirus) pHaSV1 0 1.2 ± 0.4 32Diet was limiting (ran out of food) in some cases

TABLE 4 Further bioassay of HaSV transgenic plants Four - 6 individuallarvae were fed leaf disc (50 mm²) from control or transgenic plants atone disc each per day for 4 days, before transferred to artificial dietfor a further 3 days. RNA was then extracted from the larvae andNorthern blotting with HaSV- specific probes used to verify the presenceof HaSV in the larvae. Western blot for HaSV capsid Transformed proteinin plants Mean larval Plant with (+/−) weight (mg) negative control — —positive control — — (leaf + HaSV) 3.2 pHaSV1 0 0.9 3.10 pHaSV1 0 4.83.11 pHaSV1 0 8.2Efficacy of HaSV as Atransvirus in Plants

Factors affecting the efficacy of HaSV are the viral dose required, theexpression levels achieved in plants and the leaf damage observed. Theseneed to be considered separately at this stage due to uncertainty aboutthe efficiency of HaSV assembly in plants and because larvae willcontinue feeding for about one day after receiving a toxic dose of HaSV.

I. Dose of Virus

Infection with HaSV requires neonate larvae to eat up to 10 000particles. Assuming that transgenic plants make only 1 particle percell, this means the larvae must consume up to 10 000 leaf cells.

Since a small tobacco leaf contains about one million cells, larvaecould acquire a toxic dose by consuming just 1% of the leaf.

This dose would correspond to as little as 0.000 000 5% of the solubleprotein in these cells (330×10⁻¹³ g of HaSV per leaf in 7×10⁻¹³ gsoluble plant protein per leaf).

II, Expression Levels

Assuming standard levels of 1% expression and complete incorporationinto virus particles, there should be about 10⁸ particles per cell(7×10⁻⁹ g of protein per cell over 330×10⁻¹⁹ g per HaSV particle).

However, at present only part of this protein is likely to forminfectious virus. If 1% does, then there would be 10⁶ particles percell, well above the toxic dose.

Initial results from Western blots suggest current expression at leastexceed 01.1% of soluble cell protein. Processing of the precursorprotein appears to occur to a variable extent, suggesting that particleassembly has also occurred.

The dose of infectious virus delivered by transgenic plants must bequantified by appropriately standardised bioassays.

Optimisation of the infectious virus level will be achieved by improvingvirus assembly rather than just boosting expression of components—thisrepresents a fundamental difference to the situation with toxins likeBt.

III. Leaf Damage

While as little as 1% of the leaf (and more likely far less) may besufficient to deliver a toxic dose of HaSV, larvae will keep feeding fora limited period after becoming infected. This makes it necessary todetermine the extent of leaf damage empirically.

Our initial observations were that plants making detectable levels ofHaSV capsids showed reduced susceptibility to larval feeding; this hasnot been quantified yet, and the assay was a severe one.

Consumption of leaf material by infected larvae may be estimatedindirectly using our data on larval growth and frass production, whichare approximately equal. Since neonate frass production is too low toquantify, the data were obtained from 4-day old larvae. These produce 30mg of frass over 7 days, compared to 400 mg for uninfected controls.Neonate growth and frass production may be estimated at 10% of thisfigure.

Assuming that 1 mg growth or frass—3 mg leaf material, an infectedneonate will consume about 5% of a small tobacco leaf (20 mg of a totalfresh weight of 350 mg) over seven days compared to over 60% for anuninfected control (240 mg of 350 mg).

Biosafety Considerations

It is believed that the approach of controlling pests by making aninsect virus in transgenic plants is not dangerous to the environment.This is despite our very tentative observation that some HaSVreplication is observed in protoplasts. There has been widespread debaterecently concerning the safety of protecting crops against plant virusesby inserting transgenes expressing viral proteins into the plants. Falk,B. W. and Bruening, G., 1994 (Science 263, 1395-1396) identified 3possible mechanisms which might result in the appearance of novelviruses. These mechanisms are transencapsidation, phenotypic mixing andheterologous recombination.

Transencapsidation or phenotypic mixing involving HaSV plants are notlikely to cause problem because:

the HaSV capsid gene is not acquired by the transencapsidated plantvirus genome.

such an event would yield a virus only capable of “infecting” heliothislarvae, which are not efficient vectors to enlarge the host range of aplant virus.

Heterologous recombination is not perceived as a problem because

It requires substantial sequence similarity and has only been observedwithin plant virus families. The tetraviruses are an insect specificvirus family showing minimal sequence homology to any plant RNA virus.

Even if interfamily recombination occurred, this would generate acombination of genes for which there is no precedent in either virusesinfecting both plants and insects or plants alone (HaSV would notencounter any other insect-specific virus in transgenic plants); theseviruses require functions and genes which it is physiologicallyimpossible to generate from such recombinations.

This is because:

1) the four families of insect-vectored plant viruses which replicate intheir vectors are much more complicated than HaSV, both in particlestructure and in genome organisation. All these viruses havenegative-stranded or double-stranded RNA genomes and at least 4-5 genes.

2) even the four families of insect-vectored plant viruses whichcirculate in their vectors without replicating are more complicated thanHaSV in genome organisation, number of genes and expression strategies;although they have (+)-stranded RNA genomes, they are found in adifferent virus superfamily, with replicases essentially unrelated tothat of HaSV. Their capsids are unrelated to that of HaSV and are notuncoated in the insect vectors.

All the simple, (+) stranded plant viruses which more closely resembleHaSV (which include some passively transmitted by sucking insects, iewithout entering the vector).

must have a plant cell-cell movement protein for which there is nodirect functional equivalent in HaSV.

have replicases specifically adapted to plant cells, and with minimaloverall amino acid sequence homology (under 25%) to that of HaSV.

have capsids specifically adapted for long range movement in plants andvectoring by insects without entering these or being uncoated in them;these capsid genes have no detectable sequence homology to HaSV or otherinsect capsids.

Sub-Virus

Containment Strategies

Although the expression of HaSV in transgenic plants is not consideredto present any environmental hazard, some HaSV constructs we have usedto engineer plants contain essentially suicidal versions of both RNA 1and 2. This has been achieved for RNA 2 by deleting all sequences apartfrom those directly encoding the capsid protein and demonstrating thateffective virus can still be assembled in plant cells. This alone willprevent transmission of progeny virus, since infected larvae respond asthough they had ingested normal virus, but are unable to produceinfectious progeny virus. The virus produced in plants therefore onlyinfects targets feeding on the crop plant and can neither infect otherspecies nor persist in the environment.

It is also possible to engineer subviral forms of RNA 1 which retainefficacy but do not allow production of viable progeny virus. (Forexample, remove replication signals from RNA 1).

1) Results:

The subvirus approached was tested using the following combination ofplasmids transiently expressed in protoplast and followed by bioassay asdescribed above. Bioassays weights (mg) HaSV RNAs 1 & 2 of larvae feddetected by Northern on protoplast blotting of RNA Plasmids extractsextracted from larvae 1. pDHStuR1HC + pDHVCAPB 29 ± 15 1 2. pDHStuR1HDV + pHV 57 ± 25 (—)    CAPB 3. Control: (diet only/ 85 ± 15 —   diet + protoplasts 4. pDHStuR1 HC + pDHStuR2 33 ± 28 0    HC + pDHVCAPB5. pDHStuR1 HDV + pDHStuR2 64 ± 22 —    HDV + pDHVCAPB

RNA extraction from larvae showed

(a) that larvae fed protoplasts transfected withpDHStuR1HC+pDHStuR2HC+DHVCAPB contained both RNA 1 and 2 of HaSV inintact form.

(b) that larvae fed protoplasts transfected with pDHStuR1HC+DHV CAPB(subvirus) contained a very small amount of intact HaSV RNA1 and aconsiderably greater amount of degraded RNA1.

(c) that larvae fed protoplasts transfected withpDHStuR1HDV+pDHStuR2HDV+pDHVCAPB contained no HaSVRNA with oneexception.

(d)that larvae fed protoplasts transfected with pDHStuR1HDV+pDHVCAPBcontained no HaSV RNA.

CONCLUSIONS

The HC (HaSV expression) constructs with the hairpin cassette ribozymegive infectious particles with both RNAs; the HDV expression constructsdo not under these conditions.

That the subvirus approach results in RNA1 replicating in larvae butthis RNA is degraded because it cannot be encapsidated due to missingreplicatable RNA2.

That subvirus approach gives stunting as effectively as does thecomplete virus approach under these conditions.

1. A substantially pure preparation of a Helicoverpa armigera stuntvirus, wherein the virus can infect Heliothis species and Helicoverpaspecies, and wherein the virus was deposited at the AustralianGovernment Analytical Laboratories under accession No. N92/35575.
 2. Acomposition comprising a virus according to claim 1, and anagriculturally acceptable carrier.
 3. A method of inhibiting orpreventing insect development, the method comprising exposing the insectto the virus of claim 1, where the insect is a Heliothis species orHelicoverna species.
 4. A method of inhibiting or preventing insectdevelopment, the method comprising exposing the insect to thecomposition of claim 2, where the insect is a Heliothis species orHelicoverpa species.
 5. A method of killing an insect, the methodcomprising exposing the insect to the virus of claim 1, wherein theinsect is Heliothis species or Helicoverpa species.
 6. A method ofkilling an insect, the method comprising exposing the insect to thevirus of composition of claim 2, wherein the insect is Heliothis speciesor Helicoverpa species.